Synthesis of tetracyclines and analogues thereof

ABSTRACT

The tetracycline class of antibiotics has played a major role in the treatment of infectious diseases for the past 50 years. However, the increased use of the tetracyclines in human and veterinary medicine has led to resistance among many organisms previously susceptible to tetracycline antibiotics. The modular synthesis of tetracyclines and tetracycline analogs described provides an efficient and enantioselective route to a variety of tetracycline analogs and polycyclines previously inaccessible via earlier tetracycline syntheses and semi-synthetic methods. These analogs may be used as anti-microbial agents or anti-proliferative agents in the treatment of diseases of humans or other animals.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. patent application Ser. No. 14/063,868, filedOct. 25, 2013, now U.S. Pat. No. 9,365,493, which is a continuation ofand claims priority under 35 U.S.C. §120 to U.S. patent application Ser.No. 12/778,797, filed May 12, 2010, now U.S. Pat. No. 8,598,148, whichis a divisional of and claims priority under 35 U.S.C. §120 to U.S.patent application Ser. No. 11/133,789, filed May 20, 2005, now U.S.Pat. No. 7,807,842, which claims priority under 35 U.S.C. §119(e) toU.S. provisional patent applications, U.S. Ser. No. 60/660,947, filedMar. 11, 2005, and U.S. Ser. No. 60/573,623, filed May 21, 2004, each ofwhich is incorporated herein by reference.

GOVERNMENT SUPPORT

The work described herein was supported, in part, by grants from theNational Institutes of Health (R01 AI48825) and the National ScienceFoundation (predoctoral fellowship R10964). The United States governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The tetracyclines are broad spectrum anti-microbial agents that arewidely used in human and veterinary medicine (Schappinger et al.,“Tetracyclines: Antibiotic Action, Uptake, and Resistance Mechanisms”Arch. Microbiol. 165:359-69, 1996; Mitscher, Medicinal Research Series,Vol. 9, The Chemistry of the Tetracycline Antibiotics, Marcel DekkerInc. New York, 1978). The total production of tetracyclines byfermentation or semi-synthesis is measured in the thousands of metrictons per year. The first tetracycline, chlorotetracycline (1)(Aureomycin™) was isolated from the soil bacterium Streptomycesaureofaciens by Lederle Laboratories (Wyeth-Ayerst Research) in the 1945(Duggar, Ann. N.Y. Acad. Sci. 51:177-181, 1948; Duggar, Aureomycin andPreparation of Some, U.S. Pat. No. 2,482,055, 1949; incorporated hereinby reference). Oxytetracycline (2) was isolated soon after from S.rimosus by scientists at Pfizer Laboratories (Finlay et al. Science111:85, 1950). The structures of chlorotetracycline and oxytetracyclinewere elucidated by scientists at Pfizer in collaboration with R. B.Woodward and co-workers at Harvard University (Hochstein et al. J. Am.Chem. Soc. 74:3708-3709, 1952; Hochstein et al. J. Am. Chem. Soc.75:5455-75, 1953; Stephens et al. J. Am. Chem. Soc. 74:4976-77, 1952;Stephens et al. J. Am. Chem. Soc. 76:3568-75, 1954). Tetracycline (3)was later prepared by the hydrogenolysis of chlorotetracycline and wasfound to retain the anti-microbial activity of chlorotetracycline andoxytetracycline and had increased stability (Boothe et al. J. Am. Chem.Soc. 75:4621, 1953; Conover et al. J. Am. Chem. Soc. 75:4622-23, 1953).Tetracycline was later found to be a natural product of S. aureofaciens,S. viridofaciens, and S. rimosus.

The primary tetracyclines of clinical importance today includetetracycline (3) (Boothe et al. J. Am. Chem. Soc. 75:4621, 1953),oxytetracycline (2, Terramycin™) (Finlay et al. Science 111:85, 1950),doxycycline (Stephens et al. J. Am. Chem. Soc. 85:2643, 1963), andminocycline (Martell et al. J. Med. Chem. 10:44, 1967; Martell et al. J.Med. Chem. 10:359, 1967). The tetracyclines exert their anti-microbialactivity by inhibition of bacterial protein synthesis (Bentley andO'Hanlon, Eds., Anti-Infectives: Recent Advances in Chemistry andStructure-Activity Relationships The Royal Society of Chemistry:Cambridge, UK, 1997). Most tetracyclines are bacteriostatic rather thanbactericidal (Rasmussen et al. Antimicrob. Agents Chemother. 35:2306-11,1991; Primrose and Wardlaw, Ed. “The Bacteriostatic and BacteriocidalAction of Antibiotics” Sourcebook of Experiments for the Teaching ofMicrobiology Society for General Microbiology, Academic Press Ltd.,London, 1982). It has been proposed that after tetracycline passesthrough the cytoplasmic membrane of a bacterium it chelates Mg⁺², andthis tetracycline-Mg⁺² complex binds the 30S subunit of the bacterialribosome (Goldman et al. Biochemistry 22:359-368, 1983). Binding of thecomplex to the ribosome inhibits the binding of aminoacyl-tRNAs,resulting in inhibition of protein synthesis (Wissmann et al. ForumMikrobiol. 292-99, 1998; Epe et al. EMBO J. 3:121-26, 1984).Tetracyclines have also been found to bind to the 40S subunit ofeukaryotic ribosome; however, they do not achieve sufficientconcentrations in eukaryotic cells to affect protein synthesis becausethey are not actively transported in eukaryotic cells (Epe et al. FEBSLett. 213:443-47, 1987).

Structure-activity relationships for the tetracycline antibiotics havebeen determined empirically from 50 years of semi-synthetic modificationof the parent structure (Sum et al. Curr. Pharm. Design 4:119-32, 1998).Permutations with the upper left-hand portion of the natural product,also known as the hydrophobic domain, have provided new therapeuticallyactive agents, while modifications of the polar hydrophobic domainresult in a loss of activity. However, semi-synthesis by its very naturehas limited the number of tetracycline analogs that can be prepared andstudied.

The tetracyclines are composed of four linearly fused six-membered ringswith a high density of polar functionality and stereochemicalcomplexity. In 1962, Woodward and co-workers reported the first totalsynthesis of racemic 6-desmethyl-6-deoxytetracycline (sancycline, 4),the simplest biologically active tetracycline (Conover et al. J. Am.Chem. Soc. 84:3222-24, 1962). The synthetic route was a remarkableachievement for the time and proceeded by the stepwise construction ofthe rings in a linear sequence of 22 steps (overall yield ˜0.003%). Thefirst enantioselective synthesis of (−)-tetracycline (3) from the A-ringprecursor D-glucosamine (34 steps, 0.002% overall yield) was reported byTatsuda and co-workers in 2000 (Tatsuta et al. Chem. Lett. 646-47,2000). Other approaches to the synthesis of tetracycline antibiotics,which have also proceeded by the stepwise assembly of the ABCD ringsystem beginning with D or CD precursors, include the Shemyakinsynthesis of (±)-12a-deoxy-5a,6-anhydrotetracycline (Gurevich et al.Tetrahedron Lett. 8:131, 1967; incorporated herein by reference) and theMuxfeldt synthesis of (±)-5-oxytetracycline (terramycin, 22 steps, 0.06%yield) (Muxfeldt et al. J. Am. Chem. Soc. 101:689, 1979; incorporatedherein by reference). Due to the length and poor efficiency of the fewexisting routes to tetracyclines, which were never designed forsynthetic variability, synthesis of tetracycline analogs is stilllimited.

There remains a need for a practical and efficient synthetic route totetracycline analogs, which is amenable to the rapid preparation ofspecific analogs that can be tested for improved antibacterial andpotentially antitumor activity. Such a route would allow the preparationof tetracycline analogs which have not been prepared before.

SUMMARY OF THE INVENTION

The present invention centers around novel synthetic approaches forpreparing tetracycline analogs. These synthetic approaches areparticularly useful in preparing 6-deoxytetracyclines, which are morestable towards acid and base than 6-hydroxytetracyclines. Doxycyclineand minocycline, the two most clinically important tetracyclines, aswell as tigecycline, an advanced clinical candidate, are members of the6-deoxytetracycline class.

The approaches are also useful in preparing 6-hydroxytetracyclines,pentacyclines, hexacyclines, C5-substituted tetracyclines,C5-unsubstituted tetracyclines, tetracyclines with heterocyclic D-rings,and other tetracycline analogs.

These novel synthetic approaches to tetracycline analogs involve aconvergent synthesis of the tetracycline ring system using a highlyfunctionalized chiral enone (5) as a key intermediate. The firstapproach involves the reaction of the enone with an anion formed by thedeprotonation of a toluate (6) or metallation of a benzylic halide asshown below. The deprotonation of a toluate is particularly useful inpreparing 6-deoxytetracyclines with or without a C5-substituent. Themetallation (e.g., metal-halogen exchange (e.g., lithium-halogenexchange), metal-metalloid exchange (e.g., lithium-metalloid exchange))is particularly useful in preparing 6-deoxytetracyclines with or withouta C5-substituent as well as pentacyclines. Any organometallic reagentmay be used in the cyclization process. Particularly useful reagents mayinclude lithium reagents, Grignard reagents, zero-valent metal reagents,and ate complexes. In certain embodiments, milder conditions for thecyclization reaction may be preferred.

The second approach involves reacting the enone (5) in aDiels-Alder-type reaction with a diene (7) or a benzocyclobutenol (8).

In both these approaches, the chiral enone provides the functionalized Aand B rings of the tetracycline core, and the D-ring is derived from thetoluate (6), benzylic halide, or benzocyclobutenol (8). In bringingthese two portions of the molecule together in a stereoselective mannerthe C-ring is formed. These approaches not only allow for thestereoselective and efficient synthesis of a wide variety oftetracycline analogs never before prepared, but they also allow forpreparation of tetracycline analogs in which the D-ring is replaced witha heterocycle, 5-membered ring, or other ring system. They also allowthe preparation of various pentacyclines or higher cyclines containingaromatic and non-aromatic carbocycles and heterocycles.

Through the oxidation at C6 of 6-deoxytetracycline analogs,6-oxytetracycline analogs may be prepared as shown in the scheme below:

The 6-deoxytetracycline is transformed into an aromatic naptholintermediate which undergoes spontaneous autoxidation to form thehydroperoxide. Hydrogenolysis of the hydroperoxide results in the6-oxytetracycline. This oxidation of 6-deoxytetracycline analogs can beused to prepare tetracyclines in which the D-ring is replaced with aheterocycle, 5-membered ring, or other ring system as well aspentacyclines and other polycyclines containing aromatic andnon-aromatic carbocycles and heterocycles.

The present invention not only provides synthetic methods for preparingthese tetracycline analogs but also the intermediates, including chiralenones (5), toluates (6), dienes (7), benzylic halides, andbenzocyclobutenol (8), used in these syntheses, and novel derivativesaccessed by them.

Some of the broad classes of compounds available through these newapproaches and considered to be a part of the present invention includetetracyclines and various analogs. Important subclasses of tetracyclinesinclude 6-deoxytetracyclines with or without a C5-hydroxyl group, and6-hydroxytetracyclines with or without a C5-hydroxyl group. Many of theanalogs available through these new approaches have never beensynthesized before given the limitations of semi-synthetic approachesand earlier total syntheses. For example, certain substitutions aboutthe D-ring become accessible using the present invention's novelmethodologies. In certain classes of compounds of the invention, theD-ring of the tetracyclines analog, which is usually a phenyl ring, isreplaced with a heterocyclic moiety, which may be bicyclic or tricyclic.In other classes, the D-ring is replaced with a non-aromatic ring. Thesize of the D-ring is also not limited to six-membered rings, butinstead it may be three-membered, four-membered, five-membered,seven-membered, or larger. In the case of pentacyclines, the five ringsmay or may not be linear in arrangement. Each of the D- and E-rings maybe heterocyclic or carbocyclic, may be aromatic or non-aromatic, and maycontain any number of atoms ranging from three to ten atoms. Inaddition, higher cyclines such as hexacyclines may be prepared. Incertain classes, the C-ring may not be fully formed, leading todicyclines with the A-B fused ring system intact. The compounds of theinvention include isomers, stereoisomers, enantiomers, diastereomers,tautomers, protected forms, pro-drugs, salts, and derivatives of anyparticular compound.

The present invention also includes intermediates useful in thesynthesis of compounds of the present invention. These intermediatesinclude chiral enones, toluates, benzylic halides, andbenzocyclobutenol. The intermediates includes various substituted forms,isomers, tautomers, stereoisomers, salts, and derivatives thereof.

In another aspect, the present invention provides methods of treatmentand pharmaceutical composition including the novel compounds of thepresent invention. The pharmaceutical compositions may also include apharmaceutically acceptable excipient. The methods and pharmaceuticalcompositions may be used to treat any infection including cholera,influenza, bronchitis, acne, malaria, urinary tract infections, sexuallytransmitted diseases including syphilis and gonorrhea, Legionnaires'disease, Lyme disease, Rocky Mountain spotted fever, Q fever, typhus,bubonic plague, gas gangrene, leptospirosis, whooping cough, andanthrax. In certain embodiments, the infections are caused bytetracycline-resistant organisms. In certain instances, the compounds ofthe invention exhibit anti-neoplastic or anti-proliferative activity, inwhich case the compounds may be useful in the treatment of diseases suchas cancer, autoimmune diseases, inflammatory diseases, and diabeticretinopathy. The methods and compositions may be used to treat diseasein humans and other animals including domesticated animals. Any mode ofadministration including oral and parenteral administration of thepharmaceutical composition may be used.

Given past work in the synthesis of tetracyclines, the present inventivestrategies represent a breakthrough, providing new synthetic routes totetracyclines and various analogs. The ability to prepare a wide varietyof tetracycline analogs and the use of some of these compounds in thetreatment of diseases such as cancer and infectious diseases marks anadvance not only in synthetic organic chemistry but also in medicine.The tetracycline class of antibiotics has played a major role in thetreatment of infectious diseases in human and veterinary medicine forthe past 50 years; however, with the high use of these antibiotics overmany years resistance has become a major problem. The present inventionfortunately allows for the development of tetracycline analogs withactivity against tetracycline-resistant organisms. Therefore, thedevelopments described herein will allow the tetracycline class ofantibiotics to remain part of a physician's armamentarium againstinfection diseases.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group”, as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup should be selectively removable in good yield by readilyavailable, preferably non-toxic reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.Hydroxyl protecting groups include methyl, methoxylmethyl (MOM),methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described in Protective Groups in OrganicSynthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. Furthermore, this invention is not intended to be limitedin any manner by the permissible substituents of organic compounds.Combinations of substituents and variables envisioned by this inventionare preferably those that result in the formation of stable compoundsuseful in the treatment, for example, of infectious diseases orproliferative disorders. The term “stable”, as used herein, preferablyrefers to compounds which possess stability sufficient to allowmanufacture and which maintain the integrity of the compound for asufficient period of time to be detected and preferably for a sufficientperiod of time to be useful for the purposes detailed herein.

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term“alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl”,“alkynyl”, and the like. Furthermore, as used herein, the terms “alkyl”,“alkenyl”, “alkynyl”, and the like encompass both substituted andunsubstituted groups. In certain embodiments, as used herein, “loweralkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched or unbranched) having 1-6 carbonatoms.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

The term “alkoxy”, or “thioalkyl” as used herein refers to an alkylgroup, as previously defined, attached to the parent molecule through anoxygen atom or through a sulfur atom. In certain embodiments, the alkyl,alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms. Incertain other embodiments, the alkyl, alkenyl, and alkynyl groupscontain 1-10 aliphatic carbon atoms. In yet other embodiments, thealkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4aliphatic carbon atoms. Examples of alkoxy, include but are not limitedto, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy,neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are notlimited to, methylthio, ethylthio, propylthio, isopropylthio,n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′,wherein R′ is aliphatic, as defined herein. In certain embodiments, thealiphatic group contains 1-20 aliphatic carbon atoms. In certain otherembodiments, the aliphatic group contains 1-10 aliphatic carbon atoms.In yet other embodiments, the aliphatic group employed in the inventioncontain 1-8 aliphatic carbon atoms. In still other embodiments, thealiphatic group contains 1-6 aliphatic carbon atoms. In yet otherembodiments, the aliphatic group contains 1-4 aliphatic carbon atoms.Examples of alkylamino groups include, but are not limited to,methylamino, ethylamino, n-propylamino, iso-propylamino,cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino,n-pentylamino, hexylamino, cyclohexylamino, and the like.

The term “dialkylamino” refers to a group having the structure —NRR′,wherein R and R′ are each an aliphatic group, as defined herein. R andR′ may be the same or different in an dialkyamino moiety. In certainembodiments, the aliphatic groups contains 1-20 aliphatic carbon atoms.In certain other embodiments, the aliphatic groups contains 1-10aliphatic carbon atoms. In yet other embodiments, the aliphatic groupsemployed in the invention contain 1-8 aliphatic carbon atoms. In stillother embodiments, the aliphatic groups contains 1-6 aliphatic carbonatoms. In yet other embodiments, the aliphatic groups contains 1-4aliphatic carbon atoms. Examples of dialkylamino groups include, but arenot limited to, dimethylamino, methyl ethylamino, diethylamino,methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino,di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino,di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino,di(cyclohexyl)amino, and the like. In certain embodiments, R and R′ arelinked to form a cyclic structure. The resulting cyclic structure may bearomatic or non-aromatic. Examples of cyclic diaminoalkyl groupsinclude, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl,morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

In general, the terms “aryl” and “heteroaryl”, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substituents recited for aliphatic moieties, orfor other moieties as disclosed herein, resulting in the formation of astable compound. In certain embodiments of the present invention, “aryl”refers to a mono- or bicyclic carbocyclic ring system having one or twoaromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. In certainembodiments of the present invention, the term “heteroaryl”, as usedherein, refers to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from S, O, and N; zero, one, ortwo ring atoms are additional heteroatoms independently selected from S,O, and N; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups can beunsubstituted or substituted, wherein substitution includes replacementof one, two, three, or more of the hydrogen atoms thereon independentlywith any one or more of the following moieties including, but notlimited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic, or hetercyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “heterocycloalkyl” or “heterocycle”, as used herein, refers toa non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto a benzene ring. Representative heterocycles include, but are notlimited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Incertain embodiments, a “substituted heterocycloalkyl or heterocycle”group is utilized and as used herein, refers to a heterocycloalkyl orheterocycle group, as defined above, substituted by the independentreplacement of one, two or three of the hydrogen atoms thereon with butare not limited to aliphatic; heteroaliphatic; aryl; heteroaryl;arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F;—Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples which aredescribed herein.

“Carbocycle”: The term “carbocycle”, as used herein, refers to anaromatic or non-aromatic ring in which each atom of the ring is a carbonatom.

“Independently selected”: The term “independently selected” is usedherein to indicate that the R groups can be identical or different.

“Labeled”: As used herein, the term “labeled” is intended to mean that acompound has at least one element, isotope, or chemical compoundattached to enable the detection of the compound. In general, labelstypically fall into three classes: a) isotopic labels, which may beradioactive or heavy isotopes, including, but not limited to, ²H, ³H,³²P, ³⁵S, ⁶⁷Ga, ^(99m)Tc (Tc-99m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb and ¹⁸⁶Re;b) immune labels, which may be antibodies or antigens, which may bebound to enzymes (such as horseradish peroxidase) that producedetectable agents; and c) colored, luminescent, phosphorescent, orfluorescent dyes. It will be appreciated that the labels may beincorporated into the compound at any position that does not interferewith the biological activity or characteristic of the compound that isbeing detected. In certain embodiments, hydrogen atoms in the compoundare replaced with deuterium atoms (²H) to slow the degradation ofcompound in vivo. Due to isotope effects, enzymatic degradation of thedeuterated tetracyclines may be slowed thereby increasing the half-lifeof the compound in vivo. In certain embodiments of the invention,photoaffinity labeling is utilized for the direct elucidation ofintermolecular interactions in biological systems. A variety of knownphotophores can be employed, most relying on photoconversion of diazocompounds, azides, or diazirines to nitrenes or carbenes (See, Bayley,H., Photogenerated Reagents in Biochemistry and Molecular Biology(1983), Elsevier, Amsterdam.), the entire contents of which are herebyincorporated by reference. In certain embodiments of the invention, thephotoaffinity labels employed are o-, m- and p-azidobenzoyls,substituted with one or more halogen moieties, including, but notlimited to 4-azido-2,3,5,6-tetrafluorobenzoic acid.

“Tautomers”: As used herein, the term “tautomers” are particular isomersof a compound in which a hydrogen and double bond have changed positionwith respect to the other atoms of the molecule. For a pair of tautomersto exist there must be a mechanism for interconversion. Examples oftautomers include keto-enol forms, imine-enamine forms, amide-iminoalcohol forms, amidine-aminidine forms, nitroso-oxime forms, thioketone-enethiol forms, N-nitroso-hydroxyazo forms, nitro-aci-nitroforms, and pyridione-hydroxypyridine forms.

Definitions of non-chemical terms used throughout the specificationinclude:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). A non-human animal may be a transgenic animal.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

“Effective amount”: In general, the “effective amount” of an activeagent or the microparticles refers to an amount sufficient to elicit thedesired biological response. As will be appreciated by those of ordinaryskill in this art, the effective amount of a compound of the inventionmay vary depending on such factors as the desired biological endpoint,the pharmacokinetics of the compound, the disease being treated, themode of administration, and the patient. For example, the effectiveamount of a tetracycline analog antibiotic is the amount that results ina sufficient concentration at the site of the infection to kill themicroorganism causing the infection (bacteriocidal) or to inhibit thereproduction of such microorganisms (bacteriostatic). In anotherexample, the effective amount of tetracycline analog antibiotic is theamount sufficient to reverse clinical signs and symptoms of theinfection, including fever, redness, warmth, pain, chills, cultures, andpus production.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the modular synthesis of tetracycline and tetracyclineanalogs starting from benzoic acid.

FIG. 2 depicts the total synthesis of (−)-tetracycline starting frombenzoic acid and involving an o-quinone dimethide Diels-Alder reactionbetween the chiral enone 10 and the benzocyclobutenol 11. The overallyield for the 17 step synthesis was 1.1%.

FIG. 3 is the total synthesis of (−)-doxycycline in 18 steps (overallyield 8.2%). The synthesis includes the reaction of the chiral enone 23with the anion 24 to yield the tetracycline core. The first seven stepsare identical to the first seven steps in the synthesis of(−)-tetracycline shown in FIG. 2.

FIG. 4 shows a first and second generation synthesis of isoxazole 4 usedin the synthesis of (−)-tetracycline and (−)-doxycycline as shown inFIG. 2.

FIG. 5 shows the synthesis of benzocyclobutenol 11 used in the synthesisof (−)-tetracycline as shown in FIG. 2.

FIG. 6 shows the synthesis of dicyclines. Dicyclines preserve thehydrophilic region thought to be important for the antimicrobialactivity of tetracyclines.

FIG. 7 depicts the synthesis of tricyclines via a Diels-Alder reactionwith the chiral enone 10 and a diene (41). Tricyclines preserve thehydrophobic region thought to be important for antimicrobial activity.

FIG. 8 shows the synthesis of pentacyclines.

FIG. 9 shows the synthesis of bridge pentacyclines by reacting anion 47with a chiral enone.

FIG. 10 shows five compounds that may be used as analog platforms forthe synthesis of tetracycline analogs.

FIG. 11 is a scheme showing the synthesis of a pyridone/hydroxypyridineanalog of sancycline.

FIG. 12 shows the total synthesis of 6-deoxytetracycline from benzoicacid in 14 steps (overall yield 8%). The first ten steps are identicalto the first 10 steps in the synthesis of (−)-tetracycline shown in FIG.2.

FIG. 13A shows the synthesis of a pyridine analog of sancycline,7-aza-10-deoxysancycline. FIG. 13B shows the synthesis of10-deoxysancycline.

FIGS. 14A and 14B show a number of examples of heterocyclines,tetracycline analogs, pentacyclines, and polycyclines potentiallyaccessible via the inventive method.

FIG. 15A shows the chemical structures of various tetracyclineantibiotics. (−)-Tetracycline (1) was first produced semi-synthetically,by hydrogenolysis of the fermentation product aureomycin(7-chlorotetracycline), but later was discovered to be a natural productand is now produced by fermentation (M. Nelson, W. Hillen, R. A.Greenwald, Eds., Tetracyclines in Biology, Chemistry and Medicine(Birkhauser Verlag, Boston, 2001); incorporated herein by reference).(−)-Doxycycline (2) and minocycline (3) are clinically importantnon-natural antibiotics and are both manufactured by multi-step chemicaltransformations of fermentation products (semi-synthesis) (M. Nelson, W.Hillen, R. A. Greenwald, Eds., Tetracyclines in Biology, Chemistry andMedicine (Birkhauser Verlag, Boston, 2001); incorporated herein byreference). Structures 4-6 are representative of tetracycline-likemolecules that cannot be prepared by any known semi-synthetic pathway,but which are now accessible by the convergent assembly depicted in FIG.15B. FIG. 15B depicts a generalized Michael-Dieckmann reaction sequencethat forms the C-ring of tetracyclines from the coupling of structurallyvaried carbanionic D-ring precursors with either of the AB precursors 7or 8.

FIG. 16 shows the transformation of benzoic acid in 7 steps to the keybicyclic intermediate 14. This product is then used to prepare the ABprecursor enone 7 by the 4-step sequence shown, or to enone 8, ABprecursor to 6-deoxy-5-hydroxytetracycline derivatives, by the 8-stepsequence shown.

FIG. 17 shows the synthesis of the clinically important antibiotic(−)-doxycycline (2) by the convergent coupling of the o-toluate anionderived from 18 and the AB precursor enone 8.

FIGS. 18A to 18C show the synthesis of structurally diverse6-deoxytetracyclines by coupling of structurally diverse D-ringprecursors and AB precursors 7 or 8. The number of steps and overallyields from benzoic acid are shown in parentheses below each structuresynthesized. MIC values (μg/mL) are also shown for whole-cellantibacterial testing of each analog against 5 Gram-positive and5-Gram-negative microorganisms. Corresponding MICs for tetracycline (1),a testing control, appear at bottom.

FIG. 19 shows a crystalline Michael adduct as the product of a lithiumanion and a chiral enone.

FIG. 20 shows the synthesis of a pentacycline via a Michael-Dieckmanreaction sequence.

FIGS. 21A to 21C show the synthesis of various novel tetracyclineanalogs and their corresponding D-ring precursor. These compoundsrepresent significant gaps in the tetracycline fields, likely missingfrom the literature for lack of a viable synthesis.

FIGS. 22A to 22C show alternative sequences to AB enone precursors from1S,2R-cis-dihydroxybenzoic acid.

FIGS. 23A to 23C show novel routes to AB precursors. These routes do notinvolve the microbial dihydroxylation of benzoic acid.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a strategy for the synthesis oftetracycline analogs via a convergent synthesis using as anintermediate, the highly functionalized chiral enone 9 as shown below:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthio, —NO₂, amino, alkyl amino, and dialkyl aminogroups;

P is independently selected from the group consisting of hydrogen or aprotecting group. The chiral enone 9 can be reacted with anions ofphthalides, anions of toluates, benzocyclobutenole, or dienes to yieldtetracycline analogs including heterocyclic tetracyclines, dicyclines,tricyclines, pentacyclines, heterocyclic pentacyclines, polycyclines,and heterocyclic polycyclines. These new compounds are tested foranti-microbial activity against microbes including traditionallytetracycline-sensitive organisms as well as organisms known to betetracycline-resistant. Compounds found to be bacteriocidal orbacteriostatic are used in formulating pharmaceutical for the treatmentof infections in human and veterinary medicine. The compounds are alsotested for anti-proliferative activity. Such compounds are useful in thetreatment of antiproliferative diseases including cancer,anti-inflammatory diseases, autoimmune diseases, benign neoplasms, anddiabetic retinopathy. The inventive approach to the synthesis oftetracycline analogs allows for the efficient synthesis of manycompounds never before prepared or available using earlier routes andsemi-synthetic techniques.

Compounds

Compounds of the present invention include tetracycline analogs,heterocyclic tetracycline analogs, dicyclines, tricyclines,pentacyclines, heterocylic pentatcyclines, bridged pentacyclines,heterocyclic polycyclines, bridged polycyclines, and other polycyclines.Particularly useful compounds of the present invention include thosewith biological activity. In certain embodiments, the compounds of theinvention exhibit antimicrobial activity. For example, the compound mayhave a mean inhibitory concentration, with respect to a particularbacteria, of less than 50 μg/mL, preferably less than 25 μg/mL, morepreferably less than 5 μg/mL, and most preferably less than 1 μg/mL. Forexample, infection caused by the following organisms may be treated withantimicrobial compounds of the invention:Gram-positivives—Staphylocococcus aureus, Streptococcus Group A,Streptococcus viridans, Streptococcus pneumoniae;Gram-negatives—Neisseria meningitidis, Neisseria gonorrhoeae,Haemophilus influenzae, Escherichia coli, Bacteroides fragilis, otherBacteroides; and Others—Mycoplasma pneumoniae, Treponema pallidum,Rickettsia, and Chlamydia. In other embodiments, the compounds of theinvention exhibit antiproliferative activity.

In certain embodiments, the tetracycline analogs of the presentinvention are represented by the formula:

The D-ring of 10 may include one, two, or three double bonds. In certainembodiments, the D-ring is aromatic. In other embodiments, the D-ringincludes only one double bond, and in yet other embodiments, the D-ringincludes two double bonds which may or may not be in conjugation. TheD-ring may be substituted with various groups R₇, R₆, and R₈ as definedbelow.

In 10, R₁ can be hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₁ is hydrogen, In other embodiments, R₁ is loweralkyl, alkenyl, or alkynyl. In yet other embodiments, R₁ is methyl,ethyl, n-propyl, cyclopropyl, or isopropyl. In still other embodimentsR₁ is methyl.

R₂ may be hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₂ is hydrogen. In other embodiments, R₂ ishydroxyl or a protected hydroxyl group. In certain embodiments, R₂ isalkoxy. In yet other embodiments, R₂ is a lower alkyl, alkenyl, oralkynyl group. In certain embodiments, R₁ is methyl, and R₂ is hydroxyl.In other embodiments, R₁ is methyl, and R₂ is hydrogen. In certainembodiments, R₁ and R₂ are taken together to form a carbocyclic orheterocyclic ring system spiro-linked to 10.

R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₃ is hydrogen. In other embodiments, R₃ is ahydroxyl group or a protected hydroxyl group. In yet other embodiments,R₃ is alkoxy. In still further embodiments, R₃ is lower alkyl, alkenyl,or alkynyl.

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₄ is hydrogen. In other embodiments, R₄ is ahydroxyl group or a protected hydroxyl group. In yet other embodiments,R₄ is alkoxy. In still further embodiments, R₄ is lower alkyl, alkenyl,or alkynyl. In certain embodiments, both R₃ and R₄ are hydrogen. Inother embodiments, R₃ and R₄ are taken together to form a carbocyclic orheterocyclic ring system spiro-linked to the B-ring of 10.

R₅ may be hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certainembodiments, R₅ is amino, alkylamino, or dialkylamino; preferablydimethylamino, diethylamino, methyl(ethyl)amino, dipropylamino,methyl(propyl)amino, or ethyl(propyl)amino. In other embodiments, R₅ ishydroxyl, protected hydroxyl, or alkoxy. In yet other embodiments, R₅ issulfhydryl, protected sulhydryl, or alkylthioxy.

R₇ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₇ is hydroxyl, protected hydroxyl, alkoxy, loweralkyl, lower alkenyl, lower alkynyl, or halogen.

R₆ and R₈ are absent if the dashed line between the carbon atoms whichR₆ and R₈ are attached to represents a bond, or are each selectedindependently from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, —NO₂, amino, alkyl amino, and dialkyl amino groups. Incertain embodiments, R₆ and R₈ are absent. In other embodiments, R₆ orR₈ is absent.

The variable n is an integer in the range of 0 to 8, inclusive. As willbe appreciated by one of skill in the art, when the D-ring is aromatic nis an integer between 0 and 4, preferably between 1 and 3, morepreferable between 1 and 2. In certain embodiments, when n is 2, thesubstituents R₇ are in the ortho configuration. In other embodiments,when n is 2, the substituents R₇ are in the para configuration. And inyet other embodiments, when n is 2, the substituents R₇ are in the metaconfiguration.

A dashed line in formula 10 may represent a bond or the absence of abond.

As will be appreciated by one of skill in this art, compounds of formula10 include derivatives, labeled forms, salts, pro-drugs, isomers, andtautomers thereof. Derivatives include protected forms. Salts includeany pharmaceutically acceptable salts including HCl, HBr, HI, acetate,and fatty acid (e.g., lactate, citrate, myristoleate, oleate, valerate)salts. In certain embodiments, the inventive compound exists inzwitterionic form at neutral pH with the R₅ being a protonated aminogroup and the C-3 hydroxyl group deprotonated as shown in formula 10a.

Isomers include geometric isomers, diastereomers, and enantiomers.Tautomers include both keto and enol forms of carbonyl moieties as wellas various tautomeric forms of substituted and unsubstitutedheterocycles. For example, the B-ring as shown in formula 10 includes anenol moiety as drawn, but the enol may exist as the keto form in certaincompounds as shown below in formula 10b and 10c:

Other tautomeric forms will be appreciated by one of skill in the artand will depend on the substitution pattern of the core ring structure.The formulae drawn are only given as examples and do not in any wayrepresent the full range of tautomers that may exist for a particularcompound.

Various subclasses of compounds of the formula 10 which include asubstituted or unsubstituted aromatic D-ring are shown below. Thesesubclasses include unsubstituted, monosubstituted, disubstituted, andtrisubstituted D-ring.

wherein the definitions of R₁, R₂, R₃, R₄, and R₅ are as describedabove, and R₇ is halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Incertain embodiments, R₇ is hydroxyl, protected hydroxyl, alkoxy, loweralkyl, lower alkenyl, lower alkynyl, or halogen. In other embodiments,R₇ is cyclic or acyclic, substituted or unsubstituted, branched orunbranched aliphatic; or cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic. In yet otherembodiments, R₇ is amino, alkylamino, or dialkylamino. In otherembodiments, R₇ is substituted or unsubstituted cyclic, heterocyclic,aryl, or heteroaryl. In certain embodiments, R₇ is branched orunbranched acyl.

Various subclasses of compounds of the formula 10 which include ahydroxyl group at C10 are shown:

wherein the definitions of R₁, R₂, R₃, R₄, R₅, R_(E), and R₇ are asdescribed above. In certain embodiments, the compounds are6-deoxytetracyclines as shown in the formulae below:

wherein R₂ is hydrogen, and the definitions of R₁, R₃, R₄, R₅, R_(E),and R₇ are as described above.

In another aspect of the invention, the carbocyclic D-ring oftetracycline is replaced with a heterocyclic or carbocyclic moiety asshown in formula (11):

The definitions of R₁, R₂, R₃, R₄, and R₅ are as described above forformula 10. The D-ring represented by

can be a substituted or unsubstituted aryl, heteroaryl, carbocyclic, orheterocyclic moiety, in which each occurrence of X is selected from thegroup consisting of —O—, —S—, —NR₇—, —C(R₇)₂—; n is an integer in therange of 1 to 5, inclusive; and the bonds between adjacent X moietiesare either single or double bonds. In certain embodiments,

is a polycyclic ring system such as a bicyclic or tricyclic moiety. Inother embodiments,

is a monocyclic moiety. In yet other embodiments,

is a substituted or unsubstituted heterocyclic moiety. In certainembodiments,

is not a substituted or unsubstituted phenyl ring. In other embodiments,

is a pyridinyl moiety as shown:

In another embodiment,

is selected from the group consisting of

In yet another embodiment,

is a five-membered heterocyclic ring selected from the group consistingof:

Various tetracyclines (heterocyclines) of the invention are also shownin FIGS. 14A to 14B.

Other compounds of the invention include pentacyclines of the formula:

wherein R₁, R₂, R₃, R₄, R₅, and

are as defined above. In certain embodiments, the rings of the compoundare linear. In other embodiments, the ring system is not linear. Eachoccurrence of the ring

in certain embodiments, is a monocyclic ring system. Each occurrence of

is heterocylic or carbocyclic.

is three-membered, four-membered, five-membered, six-membered, orseven-membered; preferably, five-membered or six-membered. Other classesof pentacyclines include compounds of the formulae (12), (13), and (14):

wherein R₁, R₂, R₃, R₄, R₅, and R₇ are as defined above. In formulae 12,13, and 14,

represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic,or heterocyclic moiety, in which each occurrence of X is selected fromthe group consisting of —O—, —S—, —NR₈—, —C(R₈)₂—; n is an integer inthe range of 1 to 5, inclusive; and the bonds between adjacent Xmoieties are either single or double bonds. In certain embodiments,

is a polycyclic ring system such as a bicyclic or tricyclic moiety. Inother embodiments,

is a monocyclic moiety. In other embodiments,

is a substituted or unsubstituted, aromatic or nonaromatic carbocyclicmoiety, for example a phenyl ring. In yet other embodiments,

is a substituted or unsubstituted heterocyclic moiety. In certainembodiments,

is not a substituted or unsubstituted phenyl ring. In other embodiments,

is a pyridinyl moiety as shown:

In another embodiment,

is selected from the group consisting of

In yet another embodiment,

is a five-membered heterocyclic ring selected from the group consistingof:

Various subclasses of the formula (12) include:

Various subclasses of the formula (13) include:

Various subclasses of the formula (14) include:

Various pentacyclines of the invention are also shown in FIG. 14B.

In certain embodiments, the tetracycline analogs of the presentinvention are represented by the formula:

wherein X is nitrogen, sulfur, and oxygen, and R₁, R₃, R₄, R₅, R₆, R₇,R₈, and n are defined as above with the caveat that when X is S or O, R₁is absent.

Other classes of compounds of the invention include dicyclines of theformula (15).

wherein R₃, R₄, and R₅ are as defined above. P is hydrogen or aprotecting group. R₉ is hydrogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(I); —CN; —SCN;—SR_(I); or —N(R_(I))₂; wherein each occurrence of R_(I) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certainembodiments, R₉ is hydrogen or lower (C₁-C₆) alkyl, alkenyl, or alkynyl.In other embodiments, R₉ is a vinyl group. In yet other embodiments, R₉is a substituted or unsubstituted aryl group. In still otherembodiments, R₉ is a substituted or unsubstituted heterocyclic group.

R₁₀ is cyclic or acyclic, substituted or unsubstituted, branched orunbranched aliphatic; cyclic or acyclic, substituted or unsubstituted,branched or unbranched heteroaliphatic; substituted or unsubstituted,branched or unbranched aryl; or substituted or unsubstituted, branchedor unbranched heteroaryl moiety. In certain embodiments, R₁₀ is asubstituted or unsubstituted phenyl ring. In certain embodiments, R₁₀ isa substituted or unsubstituted heterocyclic ring. In certainembodiments, R₁₀ is a substituted or unsubstituted aryl ring. In otherembodiments, R₁₀ is a lower (C₁-C₆) alkyl, alkenyl, or alkynyl group.

Methods of Synthesis

The present invention also includes all steps and methodologies used inpreparing the compounds of the invention as well as intermediates alongthe synthetic route. The present invention provides for the modularsynthesis of tetracyclines and its various analogs by joining a highlyfunctionalized chiral enone, which will become the A- and B-rings of thetetracycline core, with a molecule which will become the D-ring of thetetracycline core. The joining of these two intermediates results in theformation of the C-ring, preferably in an enantioselective manner. Thismethodology also allows for the synthesis of pentacyclines,hexacyclines, or higher ring systems as well as the incorporation ofheterocycles into the ring system. In particular, the joining of thesetwo fragments includes various nucleophilic addition reactions andcycloaddition reactions with enone (9) as described above.

The synthesis begins with the preparation of the enone (9) starting frombenzoic acid. As shown in FIG. 2, the first step of the synthesisinvolves the microbial dihydroxylation of benzoic acid using Alcaligeneseutrophus. The diol (1 in FIG. 2), which is preferably optically pure,then undergoes hydroxyl-directed epoxidation to yield the allylicepoxide (2 in FIG. 2). Protection and rearrangement of allylic epoxide 2yielded the isomeric allylic epoxide (3 in FIG. 2). The metalatedisoxazole (4 in FIG. 2) was added to the isomeric allylic epoxide toyield 5 (FIG. 2), which was subsequently metalated to close thesix-membered ring by nucleophilic attack of the epoxide. Theintermediate 6 (FIG. 2) was then rearranged, deprotected, and oxidizedto yield the chiral enone 9 (FIG. 2). As will be appreciated by one ofskill in this art, functionalization and rearrangement of intermediates6, 7, 8, and 9 in FIG. 2 will allow for the preparation of differentclass of compounds of the invention.

In one embodiment, enone (9) is reacted with an anion resulting from thedeprotonation of toluate (6). The toluate of formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and

n is an integer in the range of 0 to 3, inclusive;

R₉ is —OR_(I); —CN; —SCN; —SR_(I); or —N(R_(I))₂; wherein eachoccurrence of R_(I) is independently a hydrogen, a protecting group; acyclic or acyclic, substituted or unsubstituted aliphatic moiety; acyclic or acyclic, substituted or unsubstituted aliphaticheteroaliphatic moiety; a substituted or unsubstituted aryl moiety; or asubstituted or unsubstituted heteroaryl moiety; and

P is selected from the group consisting of hydrogen, lower (C₁-C₆) alkylgroup, an acyl group, and a protecting group;

is deprotonated under basic conditions (e.g., LDA, HMDS), and theresulting anion is reacted with an enone of formula:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthio, —NO₂, amino, alkyl amino, and dialkyl aminogroups; and

P is independently selected from the group consisting of hydrogen or aprotecting group; to form the product:

wherein R₁, R₃, R₄, R₅, R₇, P, and n are as defined above;

R₂ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Aswill be appreciated by one of skill in this art, the toluate may befurther substituted in certain embodiments. In addition, the phenyl ringof the toluate may be substituted for an aromatic heterocyclic ring suchas pyridine ring as shown in FIGS. 11 and 13A. Other examples ofcarbocyclic and heterocyclic analogs of toluate (6) include:

Other toluates are shown in FIGS. 21A to 21C. In certain embodiments,polycyclic toluates are used in the Michael-Dieckmann reaction sequenceto form pentacyclines, hexacyclines, or higher cyclines. Toluates usefulin preparing pentacyclines are exemplified by the formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

each R₇ is independently hydrogen; halogen; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl;—OR_(G); ═O; —C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G);—SO₂R_(G); —NO₂; —N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein eachoccurrence of R_(G) is independently a hydrogen, a protecting group, analiphatic moiety, a heteroaliphatic moiety, an acyl moiety; an arylmoiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio;amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthiomoiety;

represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic,or heterocyclic moiety, in which each occurrence of X is selected fromthe group consisting of —O—, —S—, —NR₈—, —C(R₈)₂—;

R₈ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(H); ═O;—C(═O)R_(H); —CO₂R_(H); —CN; —SCN; —SR_(H); —SOR_(H); —SO₂R_(H); —NO₂;—N(R_(H))₂; —NHC(O)R_(H); or —C(R_(H))₃; wherein each occurrence ofR_(H) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

n is an integer in the range of 1 to 5, inclusive; and

the bonds between adjacent X moieties are either single or double bonds;and

R₉ is selected from the group consisting of substituted or unsubstitutedaryl or heteroaryl groups.

In another embodiment, enone (9) is reacted with an anion, which isgenerated through metallation (e.g., metal-halogen exchange,metal-metalloid exchange, lithium-halogen exchange, lithium-tinexchange, etc. by reacting the toluate with the appropriate metalreagent) of a toluate of the following formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

n is an integer in the range of 0 to 3, inclusive;

R₉ is selected from the group consisting of substituted or unsubstitutedaryl or heteroaryl groups; and

Y is a halogen or Sn(R_(Y))3, wherein R_(Y) is alkyl. The aniongenerated is reacted with an enone of formula:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthio, —NO₂, amino, alkyl amino, and dialkyl aminogroups; and

P is independently selected from the group consisting of hydrogen or aprotecting group; to generate the product of formula:

wherein R₁, R₃, R₄, R₅, R₇, P, and n are as defined above; and

R₂ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

Any metal may be used in the metallation reaction to generate the metalanionic reagent to be reacted with the enone. In certain embodiments,the metal is a Group I element on the periodic chart. In otherembodiments, the metal is a Group II element on the periodic chart. Inother embodiments, the metal is a transition metal. Exemplary metalsuseful in the metallation reaction include sodium, lithium, calcium,aluminium, cadmium, copper, beryllium, arsenic, antimony, tin,magnesium, titanium, zinc, manganese, iron, cobalt, nickel, zinc,platinum, palladium, mercury, and ruthenium. In certain preferredembodiments, the metal is chosen from lithium, magnesium, titanium,zinc, and copper. In yet other embodiments, the metal is magnesium,lithium, sodium, beryllium, zinc, mercury, arsenic, antimony, or tin. Incertain particular embodiments, a lithium-halogen exchange is used. Thelithium-halogen exchange may be performed in situ in the presence of theenone. The lithium-halogen exchange may be preformed using any lithiumreagent including, for example, alkyllithium reagents, n-butyllithium,t-butyllithium, phenyl lithium, mesityl lithium, and methyllithium. Incertain embodiments, other organometallics reagents are generated andreacted with the enone. Examples include Grignard reagents, zero-valentmetal complexes, ate complexes, etc. In certain embodiments, the metalreagent is a magnesium reagent including, but not limited to, magnesiummetal, magnesium anthracene, activated magnesium turnings, etc. Incertain embodiments, the reagent is zinc-based. The reagent may begenerated in situ in the presence of the enone, or the reagent may begenerated separately and later contacted with the enone. In certainembodiments, milder conditions for the cyclization are used (e.g., azinc reagent).

As will be appreciated by one of skill in this art, the toluate may befurther substituted in certain embodiments. In addition, the phenyl ringof the toluate may be substituted for an aromatic heterocyclic ring orring system such as a pyridine ring. Examples of carbocyclic andheterocyclic analogs of toluate include:

In certain embodiments, the halogen Y is bromine. In other embodiments,Y is iodine. In yet other embodiments, Y is chloride. In certainembodiments, Y is a metalloid (e.g., tin, selenium, tellurium, etc.). Incertain embodiments, Y is —SnR₃, wherein each occurrence of R isindependently alkyl (e.g., —Sn(CH₃)₃). After the metallation reaction, Yis a metal such as lithium, magnesium, zinc, copper, antimony, sodium,etc. In certain embodiments, R₁ is hydrogen or lower alkyl (C₁-C₆). Incertain particular embodiments, R₁ is hydrogen. Other toluates are shownin FIGS. 21A to 21C.

In other embodiments, polycyclic toluates may be used to preparepentacyclines, hexacyclines, or higher cyclines. Toluates useful in thepreparation of such cyclines are of the formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

each R₇ is independently hydrogen; halogen; cyclic or acyclic,substituted or unsubstituted, branched or unbranched aliphatic; cyclicor acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; substituted or unsubstituted, branched or unbranchedacyl; substituted or unsubstituted, branched or unbranched aryl;substituted or unsubstituted, branched or unbranched heteroaryl;—OR_(G); ═O; —C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G);—SO₂R_(G); —NO₂; —N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein eachoccurrence of R_(G) is independently a hydrogen, a protecting group, analiphatic moiety, a heteroaliphatic moiety, an acyl moiety; an arylmoiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio;amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthiomoiety;

represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic,or heterocyclic moiety, in which each occurrence of X is selected fromthe group consisting of —O—, —S—, —NR₈—, —C(R₈)₂—;

R₈ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(H); ═O;—C(═O)R_(H); —CO₂R_(H); —CN; —SCN; —SR_(H); —SOR_(H); —SO₂R_(H); —NO₂;—N(R_(H))₂; —NHC(O)R_(H); or —C(R_(H))₃; wherein each occurrence ofR_(H) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

n is an integer in the range of 1 to 5, inclusive; and

the bonds between adjacent X moieties are either single or double bonds;

R₉ is selected from the group consisting of substituted or unsubstitutedaryl or heteroaryl groups; and

Y is a halogen or Sn(R_(Y))3, wherein R_(Y) is alkyl. In certainembodiments, the halogen Y is bromine. In certain embodiments, thehalogen Y is bromine. In other embodiments, Y is iodine. In yet otherembodiments, Y is chloride. In certain embodiments, Y is a metalloid(e.g., tin, selenium, tellurium, etc.). In certain embodiments, Y is—SnR₃, wherein each occurrence of R is independently alkyl (e.g.,—Sn(CH₃)₃). After the metallation reaction, Y is a metal such aslithium, magnesium, zinc, copper, sodium, mercury, antimony, etc. Incertain embodiments, R₁ is hydrogen or lower alkyl (C₁-C₆). In certainparticular embodiments, R₁ is hydrogen. In certain embodiments, R₉ isphenyl or substituted phenyl. In certain embodiments, ortho-R₇ is alkoxysuch as methoxy. In other embodiments, R₇ is hydrogen. Exemplarypolycyclic toluates include:

Compounds of the formula below with a heterocyclic C-ring:

may be prepared by Michael-Dieckmann closure of a D-ring precursorderived from the corresponding anilide, phenol, or thiophenol. Arepresentative example using anthranilic acid (i.e., anilide as thenucleophile in the Michael addition reaction) is shown below:

In another embodiment, the enone (9) is reacted with a benzocyclobutenolin an o-quinone dimethide Diels-Alder reaction. The enone of formula:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthio, —NO₂, amino, alkyl amino, and dialkyl aminogroups;

P is independently selected from the group consisting of hydrogen or aprotecting group; is reacted under suitable conditions (e.g., heat) witha benzocyclobutenol of formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

P are each selected independently from the group consisting of hydrogenor a protecting group; and

n is an integer in the range of 0 to 3, inclusive;

to form the product of formula:

wherein R₁, R₃, R₄, R₅, R₆, R₇, and P are defined as above; and

R₂ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Aswill be appreciate by one of skill in this art, the reactants may besubstituted further and still fall within the claimed invention. Forexample, the phenyl ring of the benzocyclobutenol ring may be furthersubstituted.

In another embodiment, the enone is reacted with a diene in aDiels-Alder reaction to yield a tricycline. The enone of formula:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthioxy, —NO₂, amino, alkyl amino, and dialkyl aminogroups; are as defined above; and

P is independently selected from the group consisting of hydrogen or aprotecting group; is reacted under suitable conditions (e.g., heat) witha diene of formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and

P are each selected independently from the group consisting of hydrogenand protecting groups;

to yield a protected tricycline of formula:

wherein R₂ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. Aswill be appreciated by one of skill in this art, the enone and diene maybe further substituted and still be encompassed within the presentinvention.

In yet another embodiment, the enone is reacted with an anion of aphthalide or cyano-phthalide. The enone of formula:

wherein R₃ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(C); ═O;—C(═O)R_(C); —CO₂R_(C); —CN; —SCN; —SR_(C); —SOR_(C); —SO₂R_(C); —NO₂;—N(R_(C))₂; —NHC(O)R_(C); or —C(R_(C))₃; wherein each occurrence ofR_(C) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₄ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(D); ═O;—C(═O)R_(D); —CO₂R_(D); —CN; —SCN; —SR_(D); —SOR_(D); —SO₂R_(D); —NO₂;—N(R_(D))₂; —NHC(O)R_(D); or —C(R_(D))₃; wherein each occurrence ofR_(D) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₅ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(E); —CN; —SCN;—SR_(E); or —N(R_(E))₂; wherein each occurrence of R_(E) isindependently a hydrogen, a protecting group, an aliphatic moiety, aheteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroarylmoiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino,dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₆ is selected from the group consisting of hydrogen, halogen,substituted or unsubstituted aliphatic, substituted or unsubstitutedheteroaliphatic, substituted or unsubstituted alkoxy, —OH, —CN, —SCN,—SH, alkylthio, arylthio, —NO₂, amino, alkyl amino, and dialkyl aminogroups; and

P is independently selected from the group consisting of hydrogen or aprotecting group; is reacted under basic conditions (e.g., LDA, Ph₃CLi)with the anion of the phthalide of formula:

wherein R₁ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(A); ═O;—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₇ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(G); ═O;—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

P are each selected independently from the group consisting of hydrogen,lower alkyl group, acyl group, or a protecting group; and

n is an integer in the range of 0 to 3, inclusive;

to yield a product of formula:

wherein R₂ is hydrogen; halogen; cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;substituted or unsubstituted, branched or unbranched acyl; substitutedor unsubstituted, branched or unbranched aryl; substituted orunsubstituted, branched or unbranched heteroaryl; —OR_(B); ═O;—C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B); —SOR_(B); —SO₂R_(B); —NO₂;—N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃; wherein each occurrence ofR_(B) is independently a hydrogen, a protecting group, an aliphaticmoiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; aheteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino,alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety.

The products of the above reactions are then further functionalized,reduced, oxidized, rearranged, protected, and deprotected to yield thefinal desired product. Various exemplary reactions used in the finalsyntheses of the compounds of the invention are shown in FIGS. 2, 3, 11,12, 13A, and 13B. As will be appreciated by one of skill in the art,various isolation and purification techniques including flashchromatography, crystallization, distillation, HPLC, thin layerchromatography, extraction, filtration, etc. may be used in the courseof synthesizing compounds of the invention. These techniques may be usedin the preparation or purification of intermediates, reagents, products,starting materials, or solvents.

Pharmaceutical Compositions

This invention also provides a pharmaceutical preparation comprising atleast one of the compounds as described above and herein, or apharmaceutically acceptable derivative thereof, which compounds inhibitthe growth of or kill microorganisms, and, in certain embodiments ofspecial interest are inhibit the growth of or killtetracycline-resistant organisms including chlortetracycline-resistantorganisms, oxytetracycline-resistant organisms, demeclocycline-resistantorganisms, doxycycline-resistant organisms, minocycline-resistantorganisms, or any organisms resistant to antibiotics of the tetracyclineclass used in human or veterinary medicine. In other embodiments, thecompounds show cytostatic or cytotoxic activity against neoplastic cellssuch as cancer cells. In yet other embodiments, the compounds inhibitthe growth of or kill rapidly dividing cells such as stimulatedinflammatory cells.

As discussed above, the present invention provides novel compoundshaving antimicrobial and antiproliferative activity, and thus theinventive compounds are useful for the treatment of a variety of medicalconditions including infectious diseases, cancer, autoimmune diseases,inflammatory diseases, and diabetic retinopathy. Accordingly, in anotheraspect of the present invention, pharmaceutical compositions areprovided, wherein these compositions comprise any one of the compoundsas described herein, and optionally comprise a pharmaceuticallyacceptable carrier. In certain embodiments, these compositionsoptionally further comprise one or more additional therapeutic agents,e.g., another anti-microbial agent or another anti-proliferative agent.In other embodiments, these compositions further comprise ananti-inflammatory agent such as aspirin, ibuprofen, acetaminophen, etc.,pain reliever, or anti-pyretic.

It will also be appreciated that certain of the compounds of the presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or any other adduct or derivative which uponadministration to a patient in need is capable of providing, directly orindirectly, a compound as otherwise described herein, or a metabolite orresidue thereof, e.g., a prodrug.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19, 1977; incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting the free base functionality with a suitable organic orinorganic acid. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate, and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters which hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates. In certainembodiments, the esters are cleaved by enzymes such as esterases.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the anti-cancercompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;Cremophor; Solutol; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

Uses of Compounds and Pharmaceutical Compositions

The invention further provides a method of treating infections andinhibiting tumor growth. The method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it.

The compounds and pharmaceutical compositions of the present inventionmay be used in treating or preventing any disease or conditionsincluding infections (e.g., skin infections, GI infection, urinary tractinfections, genito-urinary infections, systemic infections),proliferative diseases (e.g., cancer), and autoimmune diseases (e.g.,rheumatoid arthritis, lupus). The compounds and pharmaceuticalcompositions may be administered to animals, preferably mammals (e.g.,domesticated animals, cats, dogs, mice, rats), and more preferablyhumans. Any method of administration may be used to deliver the compoundof pharmaceutical compositions to the animal. In certain embodiments,the compound or pharmaceutical composition is administered orally. Inother embodiments, the compound or pharmaceutical composition isadministered parenterally.

In yet another aspect, according to the methods of treatment of thepresent invention, bacteria are killed, or their growth is inhibited bycontacting the bacteria with an inventive compound or composition, asdescribed herein. Thus, in still another aspect of the invention, amethod for the treatment of infection is provided comprisingadministering a therapeutically effective amount of an inventivecompound, or a pharmaceutical composition comprising an inventivecompound to a subject in need thereof, in such amounts and for such timeas is necessary to achieve the desired result. In certain embodiments ofthe present invention a “therapeutically effective amount” of theinventive compound or pharmaceutical composition is that amounteffective for killing or inhibiting the growth of bacteria. Thecompounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for killing or inhibiting the growth ofbacteria. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the infection, the particular compound, its mode ofadministration, its mode of activity, and the like. The compounds of theinvention are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the compounds and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient or organism will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionsof this invention can be administered to humans and other animalsorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, or drops),bucally, as an oral or nasal spray, or the like, depending on theseverity of the infection being treated. In certain embodiments, thecompounds of the invention may be administered orally or parenterally atdosage levels sufficient to deliver from about 0.001 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 10 mg/kg, and more preferably from about 1 mg/kg to about 25mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic effect. The desired dosage may bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks, orevery four weeks. In certain embodiments, the desired dosage may bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations).

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active compounds, the liquid dosage forms may contain inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the compounds of theinvention are mixed with solubilizing agents such an Cremophor,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies, that is, the compounds and pharmaceutical compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with anotheranticancer agent), or they may achieve different effects (e.g., controlof any adverse effects).

In still another aspect, the present invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention, and in certain embodiments, includes an additional approvedtherapeutic agent for use as a combination therapy. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1—Synthesis of (−)-Tetracycline

General Procedures.

All reactions were performed in flame-dried round bottomed or modifiedSchlenk (Kjeldahl shape) flasks fitted with rubber septa under apositive pressure of argon, unless otherwise noted. Air- andmoisture-sensitive liquids and solutions were transferred via syringe orstainless steel cannula. Where necessary (so noted), solutions weredeoxygenated by alternative freeze (liquid nitrogen)/evacuation/thawcycles (≧three iterations). Organic solutions were concentrated byrotary evaporation at ˜25 Torr (house vacuum). Flash columnchromatography was performed on silica gel (60 Å, standard grade) asdescribed by Still et al. (Still, W. C.; Kahn, M.; Mitra, A. J. Org.Chem. 1978, 43, 2923-2925; incorporated herein by reference). Analyticalthin-layer chromatography was performed using glass plates pre-coatedwith 0.25 mm 230-400 mesh silica gel impregnated with a fluorescentindicator (254 nm). Thin layer chromatography plates were visualized byexposure to ultraviolet light and/or exposure to ceric ammoniummolybdate or an acidic solution of p-anisaldehyde followed by heating ona hot plate.

Materials.

Commercial reagents and solvents were used as received with thefollowing exceptions. Chlorotrimethylsilane, triethylamine,diisopropylamine, 2,2,6,6-tetramethylpiperidine, N,N,N′,N′-tetramethylethylenediamine, DMPU, HMPA, andN,N-diisopropylethylamine were distilled from calcium hydride underdinitrogen atmosphere. Benzene, dichloromethane, ethyl ether, methanol,pyridine, tetrahydrofuran, hexane, acetonitrile, N,N-dimethylformamide,and toluene were purified by the method of Pangborn et al. (Pangborn, A.B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J.Organometallics 1996, 15, 1518-1520; incorporated herein by reference).The molarity of n-butyllithium, s-butyllithium, and t-butyllithium weredetermined by titration with a tetrahydrofuran solution of 2-butanolusing triphenylmethane as an indicator (Duhamel, L.; Palquevent, J.-C.J. Org. Chem. 1979, 44, 3404-3405; incorporated herein by reference).

Instrumentation.

Proton nuclear magnetic resonance (¹H NMR) spectra and carbon nuclearmagnetic resonance (¹³C NMR) were recorded with Varian Unity/Inova 600(600 MHz), Varian Unity/Inova 500 (500 MHz/125 MHz), or Varian Mercury400 (400 MHz/100 MHz) NMR spectrometers. Chemical shifts for protons arereported in parts per million scale (δ scale) downfield fromtetramethylsilane and are referenced to residual protium in the NMRsolvents (CHCl₃: δ7.26, C₆D₅H: δ 7.15, D₂HCOD: δ 3.31, CDHCl₂: δ 5.32,(CD₂H)CD₃SO: δ 2.49). Chemical shifts for carbon are reported in partsper million (δ scale) downfield from tetramethylsilane and arereferenced to the carbon resonances of the solvent (CDCl₃: δ 77.0, C₆D₆:δ 128.0, D₃COD: δ 44.9, CD₂Cl₂: δ 53.8, (CD₃)₂SO: δ 39.5). Data arerepresented as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), integration,coupling constant in Hz, and assignment. Infrared (IR) spectra wereobtained using a Perkin-Elmer 1600 FT-IR spectrophotometer referenced toa polystyrene standard. Data are represented as follows: frequency ofthe absorption (cm⁻¹), intensity of absorption (s=strong, sb=strongbroad, m=medium, w=weak, br=broad), and assignment (where appropriate).Optical rotations were determined on a JASCO DIP-370 digital polarimeterequipped with a sodium lamp source using a 200 μL or 2-mL solution cell.High resolution mass spectra were obtained at the Harvard UniversityMass Spectrometry Facilities.

Microbial Dihydroxylation Product DRS1:

Preparation of Glycerol Stock Solutions

Alcaligenes eutrophus B9 cells (lyophilized powder, 20 mg, generouslysupplied by Prof. George D. Hegeman (Indiana University); Reiner, A. M.;Hegeman, G. D. Biochemistry 1971, 10, 2530.) were suspended in nutrientbroth (5 mL, prepared by dissolving 8 g of Difco Bacto© Nutrient Brothin 1 L of nanopure water followed by sterilization in an autoclave at125° C.) in a 20-mL sterile culture tube. Aqueous sodium succinatesolution (16.7 μL of a 2.5 M aqueous solution, 5 mM final concentration)was added, and the culture tube was shaken at 250 rpm at 30° C. untilcell growth became apparent (3 d). An aliquot (250 μL) of the cellularsuspension was then transferred to 5 mL of Hutner's mineral base medium(HMB, see paragraph below) containing sodium succinate (16.7 μL of a 2.5M aqueous solution, 5 mM final concentration) in a 20-mL sterile culturetube. The culture tube was shaken at 250 rpm for 2 d at 30° C.,whereupon an aliquot (250 μL) of the fermentation solution wassubcultured in a sterile Erlenmeyer flask containing 50 mL of HMB andaqueous sodium succinate solution (167 μL of a 2.5 M solution, 5 mMfinal concentration). The flask was shaken at 250 rpm for 24 h at 30° C.The resulting solution was used directly for the preparation of glycerolstock solutions. Thus, a portion of the subcultured cellular suspension(5 mL) was diluted with an equal volume of sterile glycerol, and theresulting solution was divided equally into ten 2-mL sterile Eppendorftubes. The individual stock solutions were then stored at −80° C.

Hutner's Mineral Base Medium

Hutner's mineral base medium (HMB) was prepared as follows. Solidpotassium hydroxide (400 mg) was dissolved in 500 mL of nanopure waterin a 2-L Erlenmeyer flask. Nitrilotriacetic acid (200 mg), magnesiumsulfate (283 mg), calcium chloride dihydrate (67 mg), ammonium molybdate(0.2 mg), iron (II) sulfate (2.0 mg), Hutner's Metals 44 solution (1 mL,see paragraph below), ammonium sulfate (1.0 g), potassium dihydrogenphosphate (2.72 g) and sodium monohydrogen phosphate heptahydrate (5.36g) were added sequentially. The solution was diluted to a total volumeof 1 L and the pH was adjusted to 6.8 with concentrated hydrochloricacid. The medium was sterilized by filtration or by heating in anautoclave.

Hutner's Metals 44 solution was prepared as follows. Concentratedsulfuric acid (100 μL) was added to nanopure water (50 mL) in a 250-mLErlenmeyer flask. Solid EDTA (0.50 g), zinc sulfate heptahydrate (2.20g), iron (II) sulfate heptahydrate (1.0 g), copper (I) sulfate (0.39 g),cobalt (II) nitrate hexahydrate (50 mg) and sodium tetraboratedecahydrate (36 mg) were then added in sequence, followed by 50 mL ofnanopure water.

Cellular Dihydroxylation of Sodium Benzoate

A sterile pipette tip was streaked across the surface of a frozenglycerol stock solution to produce small shards (ca. 10 mg). The frozenshards were added to a sterile 125 mL Erlenmeyer flask containing HMB(25 mL) and aqueous sodium succinate solution (140 μL of a 1.5 Msolution, 5 mM final concentration). The flask was shaken at 250 rpm for2 days at 30° C. An aliquot (10 mL) of the white, heterogeneous solutionwas transferred using a sterile pipette to a mammalian cell growth jarcontaining HMB (6 L) and aqueous sodium succinate solution (20 mL of a1.5 M solution, 5 mM final concentration). The jar was warmed on a hotplate to an internal temperature of 30° C.; cotton-filtered air wassparged through the medium. After 2 days, the white, heterogeneoussolution was treated with aqueous sodium benzoate solution (18 mL of a1.0 M solution) and aqueous sodium succinate solution (10 mL of a 1.5 Msolution), inducing dihydroxylation. The resulting mixture was aeratedvigorously for 6 hours at an internal temperature of 30° C. Afterinduction, sufficient aqueous sodium benzoate solution (24 to 48 mL of a1.0 M solution, depending on the rate of consumption) was added hourlyto maintain a concentration of 10-20 mM (determined by UV absorbance at225 nm). Aqueous sodium succinate solution (10 mL of a 1.5 M solution)was added every fourth hour. These additions proceeded over 18 hours,then the solution was aerated overnight at an internal temperature of30° C., to ensure complete conversion. The fermentation broth wascentrifuged, in portions, at 6000 rpm (Sorvall GS-3 rotor, modelSLA-3000) to remove cellular material. The supernatant was concentratedto a volume of 400 mL using a rotary evaporator (bath temperature <45°C.). The concentrate was cooled to 0° C. and then acidified to pH 3.0using concentrated aqueous hydrochloric acid. The acidified aqueoussolution was extracted repeatedly with ethyl acetate (8×500 mL, 4×800mL, 8×1 L). The ethyl acetate extracts were dried over sodium sulfatebefore concentration, using a rotary evaporator (bath temperature <45°C.), providing a pale yellow solid residue. Trituration of the residuewith dichloromethane (2×200 mL) followed by drying in vacuo affordedpure (1S,2R)-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylic acid (DRS1)as a white powder mp 95-96° C. dec (38 g, 74%, [α]_(D) −114.8 (c 0.5 inEtOH), lit., [α]_(D) −106 (c 0.5 in EtOH) Jenkins, G. N.; Ribbons, D.W.; Widdowson, D. A.; Slawin, A. M. Z.; Williams, D. J. J. Chem. Soc.Perkin Trans. 1 1995, 2647.).

Epoxide DRS2:

m-Chloroperoxybenzoic acid (mCPBA was purified as follows: 50 g of 77%mCPBA (Alrich) was dissolved in benzene (1 L), the benzene solution wasthen washed with pH 7.4 phosphate buffer (3×1 L) and dried over Na₂SO₄for 3 hours and concentrated (<40° C., thermal detonation hazard) toprovide pure mCPBA as a white solid; 10.7 g, 62.3 mmol, 1.2 equiv) wasadded in three equal portions over 30 min. to a suspension of themicrobial dihydroxylation product DRS1 (8.10 g, 51.9 mmol, 1.0 equiv) inethyl acetate (400 mL) at 23° C. The heterogeneous solution was stirredfor 10 h, then was diluted with benzene (80 mL) and stirred for 1 h Thesupernatant was decanted and the solid residue was triturated withbenzene (2×15 mL). The resulting pasty solid was dried in vacuo toprovide the epoxide DRS2 as an amorphous white powder (7.36 g, 83%).

mp 87-91° C.; ¹H NMR (400 MHz, CD₃OD) δ 6.23 (dd, 1H, J=9.6, 3.9 Hz,═CHC(OCH)), 5.92 (dd, 1H, J=9.6, 1.9 Hz, ═CHC(CO₂H)), 4.40 (d, 1H, J=1.3Hz, CHOH), 3.58 (dd, 1H, J=4.4, 1.3 Hz, CHCHOH), 3.49 (m, 1H, ═CCHO);¹³C NMR (100 MHz, CD₃OD) δ 175.8, 135.1, 128.8, 75.4, 70.9, 57.5, 50.3;FTIR (neat), cm⁻¹ 3381 (s, OH), 1738 (s, C═O), 1608 (m), 1255 (m), 1230(m), 1084 (m, C—O); HRMS (CI) m/z calcd for (C₇H₈O₅+NH₄)⁺ 190.0715.found 190.0707.

Epoxide DJB1:

A solution of trimethylsilyldiazomethane in hexanes (2.0 M, 25.5 mL,51.0 mmol, 1.2 equiv) was added to a solution of the epoxide DRS2 (7.36g, 42.8 mmol, 1.0 equiv) in methanol-benzene (1:3, 160 mL) at 23° C.Extensive gas evolution was observed upon addition. The yellow solutionwas stirred for 5 min, then was concentrated, affording a light yellowsolid. The solid was dried by azeotropic distillation from benzene (2×25mL), and the dried solid was suspended in dichloromethane (200 mL).Triethylamine (20.8 ml, 149 mmol, 3.5 equiv) and tert-butyldimethylsilyltrifluoromethanesulfonate (29.4 ml, 128 mmol, 3.0 equiv) were then addedin sequence, providing a homogeneous solution. The reaction solution wasstirred at 23° C. for 30 min. An aqueous potassium phosphate buffersolution (pH 7.0, 0.2 M, 300 mL) was added followed by dichloromethane(100 ml). The organic phase was separated and dried over anhydroussodium sulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a brown oil. The product was purified by flashcolumn chromatography (5:95 ethyl acetate-hexanes), affording theepoxide DJB1 as a light yellow oil (12.4 g, 70% over 2 steps). R_(f)0.50 (1:4 ethyl acetate-hexanes); ¹H NMR (400 MHz, CDCl₃) δ 5.95 (dd,1H, J=9.8, 3.4 Hz, ═CHCOTBS), 5.89 (ddd, 1H, J=9.8, 2.9, 1.5 Hz,═CHCHOCCO₂), 4.63 (d, 1H, J=3.9 Hz, O₂CCCHOTBS), 4.42 (m, 1H, ═CCHOTBS),3.78 (s, 3H, OCH₃), 3.31 (d, 1H, J=2.0 Hz, CHOCCO₂), 0.90 (s, 9H,C(CH₃)₃), 0.89 (s, 9H, C(CH₃)₃), 0.09 (s, 3H, SiCH₃), 0.08 (s, 6H,SiCH₃), 0.07 (s, 3H, SiCH₃); ¹³C NMR (100 MHz, CDCl₃) δ 170.2, 138.7,122.6, 69.3, 68.4, 59.7, 52.5, 52.0, 25.9, 25.7, 18.3, 18.2, −4.18,−4.27, −4.45, −5.21; FTIR (neat), cm⁻¹ 1759 (m, C═O), 1736 (s, C═O),1473 (m), 1256 (w), 1253 (s), 1150 (s, C—O), 1111 (m, C—O), 1057 (s,C—O), 940 (m); HRMS (ES) m/z calcd for (C₂₀H₃₈O₅Si₂)⁺ 414.2258. found414.2239.

Isoxazole MGC2 (Method A):

Triethylamine (37.5 mL, 0.269 mol, 1.15 equiv),4-(dimethylamino)pyridine (289 mg, 2.34 mmol, 0.01 equiv), andmethanesulfonyl chloride (20.8 mL, 0.269 mol, 1.15 equiv) were added insequence to a solution of the alcohol MGC1 (prepared in two steps fromcommercially available methyl 3-hydroxy-5-isoxazolecarboxylate aspreviously reported by: Reiss, R.; Schön, M.; Laschat, S.; Jäger, V.Eur. J. Org. Chem. 1998, 473-479.) (48.0 g, 0.234 mol, 1.0 equiv) indichloromethane (450 mL) at 0° C. The reaction mixture was stirred at 0°C. for 2.5 h, then was concentrated, affording an orange oil. Chilleddimethylamine (condensed using a cold finger with dry ice/acetone, 26.2mL, 0.480 mol, 2.0 equiv) was added to a mixture of the orange oilprepared above and N,N-dimethylformamide (150 mL) at 0° C., providing ahomogenous solution. The solution was stirred at 0° C. for 2 h, then wasallowed to warm to 23° C.; stirring was continued at that temperaturefor 24 h. The solution was partitioned between saturated aqueous sodiumbicarbonate solution-brine (2:1, 300 mL) and ethyl acetate-hexanes (1:1,500 mL). The organic phase was separated and washed with brine (2×200mL), and dried over anhydrous sodium sulfate The dried solution wasfiltered and the filtrate was concentrated, furnishing a brown residue.The product was purified by flash column chromatography (1:4 to 1:1ethyl acetate-hexanes), affording the isoxazole MGC2 as a light yellowoil (40.1 g, 74%). R_(f) 0.34 (1:1 ethyl acetate-hexanes); ¹H NMR (500MHz, CDCl₃) δ 7.43-7.31 (m, 5H, ArH), 5.82 (s, 1H, ═CH), 5.23 (s, 2H,OCH₂Ar), 3.48 (s, 2H, CH₂N(CH₃)₂), 2.27 (s, 6H, N(CH₃)₂); ¹³C NMR (125MHz, CDCl₃) δ 171.9, 171.2, 136.1, 128.8, 128.5, 128.7, 94.8, 71.7,55.1, 45.3; FTIR (neat), cm⁻¹ 2950 (s, CH), 1615 (s), 1494 (s), 1452(s), 1136 (m); HRMS (ES) m/z calcd for (C₁₃H₁₆N₂O₂)⁺ 232.1212. found232.1220.

Isoxazole MGC4:

Chilled dimethylamine (condensed into a reaction vessel submerged in a0° C. bath using a cold finger with dry ice/acetone, 106 mL, 1.94 mol,2.2 equiv) was added dropwise via cannula to a solution of the isoxazoleMGC3 (prepared in two steps from glyoxylic acid as reported by:Pevarello, P.; Varasi, M. Synth. Commun. 1992, 22, 1939.) (174 g, 0.884mol, 1.0 equiv) in acetonitrile (2 L) at 0° C. The reaction mixture wasstirred at 0° C. for 2 h, then the cooling bath was removed. Thereaction mixture was allowed to warm to 23° C.; stirring was continuedat that temperature for 8 h. The mixture was partitioned betweenbrine-saturated aqueous sodium bicarbonate solution (1:1, 1.5 L) andethyl acetate (1.5 L). The organic phase was separated and the aqueousphase was further extracted with ethyl acetate (3×400 mL). The organicphases were combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated to a volume of500 mL, resulting in the formation of a white precipitate. Theconcentrate was filtered and the filtrate was concentrated, providingthe isoxazole MGC4 as an orange oil (143 g, 79%). An analytical samplewas prepared by flash column chromatography (1:9 to 2:8 ethylacetate-hexanes), affording the isoxazole MGC4 as a light yellow oil.

R_(f) 0.30 (1:4 ethyl acetate-hexanes); ¹H NMR (300 MHz, CDCl₃) δ 6.26(s, 1H, vinyl), 3.63 (s, 2H, CH₂N(CH₃)₂), 2.30 (s, 6H, N(CH₃)₂); ¹³C NMR(100 MHz, CDCl₃) δ 172.1, 140.5, 106.8, 54.5, 45.3; FTIR (neat), cm⁻¹3137 (w), 2945 (m), 2825 (m), 2778 (m), 1590 (s), 1455 (m), 1361 (m),1338 (s), 1281 (s), 1041 (m); HRMS (ES) m/z calcd for (C₆H₉BrN₂O+H)⁺204.9976. found 204.9969.

Isoxazole MGC2 (Method B):

Sodium metal (32.63 g, 1.42 mol, 2.03 equiv) was added portionwise over8 h to benzyl alcohol (1 L) at 23° C. The resulting mixture was stirredvigorously for 24 h, then was transferred via large bore cannula to theneat isoxazole MGC4 (143 g, 0.700 mol, 1.0 equiv) at 23° C. Theresulting light brown mixture was placed in an oil bath preheated to120° C. and was stirred for 20 h at that temperature. Ethyl acetate (2L) was added to the cooled reaction mixture and stirring was continuedfor 15 min. Aqueous hydrochloric acid (1.0 M, 2 L) was added and theaqueous phase was separated. The organic phase was further extractedwith two 300-mL portions of 1.0 M aqueous hydrochloric acid. The aqueousphases were combined and the pH adjusted to 9 by slow addition ofaqueous sodium hydroxide (6.0 M, approx. 350 mL). The resulting mixturewas extracted with dichloromethane (3×500 mL). The organic extracts werecombined and dried over anhydrous sodium sulfate. The dried solution wasfiltered and the filtrate was concentrated, yielding the isoxazole MGC2as a yellow oil (102 g, 63%). An analytical sample was prepared by flashcolumn chromatography (3:7 ethyl acetate-hexanes, then 5:95 methanol inethyl acetate), affording the isoxazole MGC2 as a light yellow oil(spectroscopic data was identical to that obtained for material preparedby Method A).

Ketone MGC5:

A solution of n-butyllithium in hexanes (2.47 M, 16.0 mL, 39.5 mmol, 1.0equiv) was added to a solution of the isoxazole MGC2 (9.16 g, 39.5 mmol,1.0 equiv) in tetrahydrofuran (150 mL) at −78° C. The resultingrust-colored solution was stirred at −78° C. for 1 h whereupon asolution of the methyl ester DJB1 (9.82 g, 23.7 mmol, 0.6 equiv) intetrahydrofuran (6 mL) was added dropwise via cannula. The transfer wasquantitated with two 1-mL portions of tetrahydrofuran. The resultingbrown solution was stirred at −78° C. for 1 h, then an aqueous potassiumphosphate buffer solution (pH 7.0, 0.2 M, 250 mL) was added. Thebiphasic mixture was allowed to warm to 23° C., then was extracted withdichloromethane (2×300 mL). The organic extracts were combined and driedover anhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a yellow oil. The product waspurified by flash column chromatography (1:9 to 1:3 ethylacetate-hexanes), affording the ketone MGC5 as a light yellow solid(10.6 g, 73%).

R_(f) 0.59 (1:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ7.44-7.35 (m, 5H, ArH), 5.90 (ddd, 1H, J=9.8, 5.9, 2.0 Hz, ═CHCHOSi),5.82 (dd, 1H, J=9.8, 3.4 Hz, ═CHCHOCC), 5.31 (m, 2H, OCH₂Ar), 4.58 (d,1H, J=4.2 Hz, (O)CCCHOSi), 4.27 (m, 1H, ═CHCHOSi), 3.94 (d, 1H, J=15.6Hz, CHH′N), 3.77 (d, 1H, J=15.6 Hz, CHH′N), 3.17 (dd, 1H, J=3.4, 1.5 Hz,HCOCC(O)), 2.35 (s, 6H, N(CH₃)₂), 0.89 (s, 9H, C(CH₃)₃), 0.83 (s, 9H,C(CH₃)₃), 0.06 (s, 3H, SiCH₃), 0.05 (s, 3H, SiCH₃), 0.04 (s, 3H, SiCH₃),−0.07 (s, 3H, SiCH₃); ¹³C NMR (125 MHz, CDCl₃) δ 191.8, 176.3, 168.9,136.5, 135.5, 128.8, 128.7, 125.0, 106.9, 72.4, 69.6, 67.8, 67.4, 55.3,52.6, 45.9, 26.2, 26.0, 18.5, 18.3, −3.1, −3.8, −3.8, −5.1; FTIR (neat),cm⁻¹ 2952 (s, CH), 1682 (s, C═O), 1594 (s), 1502 (s), 1456 (m), 1097 (s,C—O), 774 (s); HRMS (FAB) m/z calcd for (C₃₂H₅₀N₂O₆Si₂+Na)⁺ 637.3105.found 637.3097.

Ketones MGC6 and MGC7:

Solid lithium trifluoromethanesulfonate (76.0 mg, 0.490 mmol, 0.05equiv) was added to a solution of the ketone MGC5 (6.02 g, 9.80 mmol,1.0 equiv) in toluene (500 mL) at 23° C. The resulting heterogeneouslight yellow mixture was placed in an oil bath preheated to 65° C. andwas stirred at that temperature for 3 h. The reaction mixture was cooledto 23° C. and was filtered. The solids were washed with toluene (50 mL)and the filtrate was concentrated, providing a yellow oil. The oil wascovered with dichloromethane-trifluoroacetic acid (10:1, 165 mL) and theresulting mixture was stirred at 23° C. for 18 h. Aqueous sodiumbicarbonate solution (300 mL) was added and extensive gas evolution wasobserved upon addition. The biphasic mixture was extracted with diethylether (4×300 mL) and the organic extracts were combined and dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a brown oil. The product waspurified by flash column chromatography (1:9 to 1:5 ethylacetate-hexanes), affording the ketone MGC6 as a white foam (3.20 g,62%) and the ketone MGC7 as a viscous yellow oil (1.68 g, 28%).

Ketone MGC6:

R_(f) 0.52 (1:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.45(m, 2H, ArH), 7.36-7.30 (m, 3H, ArH), 5.96 (bs, 1H, ═CH), 5.45 (bs, 1H,═CH), 5.32 (m, 2H, OCHH′Ar), 5.33 (bs, 1H, CHOSi), 4.15 (d, 1H, J=8.8Hz, CHOSi), 3.59 (d, 1H, J=3.9 Hz, CHN(CH₃)₂), 3.34 (bs, 1H, C₃CH), 2.57(bs, 1H, OH), 2.39 (s, 6H, N(CH₃)₂), 0.90 (s, 9H, C(CH₃)₃), 0.16 (s, 3H,SiCH₃), 0.11 (s, 3H, SiCH₃); ¹³C NMR (100 MHz, C₆D₆) δ 189.2, 178.3,168.6, 135.3, 128.5, 128.4, 128.3, 125.4, 106.4, 79.8, 72.3, 72.2, 67.1,63.6, 42.9, 26.1, 18.5, −4.0, −4.8; FTIR (neat), cm⁻¹ 3549 (bs, OH),3455 (bs, OH), 2942 (s, CH), 1698 (s, C═O), 1617 (m), 1508 (s), 1032 (s,C—O), 906 (s); HRMS (ES) m/z calcd for (C₂₆H₃₆N₂O₆Si+H)⁺ 501.2421. found501.2422.

Ketone MGC7:

R_(f) 0.64 (1:5 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.50(d, 2H, J=1.5 Hz, ArH), 7.40-7.32 (m, 3H, ArH), 5.94 (dd, 1H, J=9.7, 6.4Hz, ═CHCHCHOSi), 5.76 (d, 1H, J=9.7 Hz, ═CHCOH), 5.37 (d, 1H, J=12.2 Hz,OCHH′Ph), 5.32 (d, 1H, J=12.2 Hz, OCHH′Ph), 4.09 (d, 1H, J=2.9 Hz,HOCCHOSi), 4.03 (s, 1H, OH), 3.88 (m, 1H, NCHCHCHOSi), 3.74 (d, 1H,J=3.9 Hz, (CH₃)₂NCH), 2.46 (s, 6H, N(CH₃)₂), 0.91 (s, 9H, C(CH₃)₃), 0.87(s, 9H, C(CH₃)₃), 0.06 (s, 3H, SiCH₃), 0.05 (s, 3H, SiCH₃), 0.04 (s, 3H,SiCH₃), 0.03 (s, 3H, SiCH₃); ¹³C NMR (125 MHz, CDCl₃) δ 194.9, 173.9,170.5, 135.8, 132.6, 128.8, 128.5, 128.3, 127.9, 106.2, 81.6, 74.8,72.0, 71.7, 69.5, 44.6, 43.2, 26.1, 25.9, 18.7, 18.2, −3.6, −4.1, −4.3,−4.3; FTIR (neat), cm⁻¹ 3461 (bs, OH), 2940 (s, CH), 1693 (s, C═O), 1663(s), 1647 (m), 1503 (m), 1080 (s, C—O), 774 (s); HRMS (ES) m/z calcd for(C₃₂H₅₀N₂O₆Si₂+H)⁺ 615.3285. found 615.3282.

Alkene DRS3:

Diethyl azodicarboxylate (472 μL, 3.00 mmol, 3.0 equiv) was added to asolution of the ketone MGC6 (500 mg, 1.00 mmol, 1.0 equiv) andtriphenylphosphine (789 mg, 3.00 mmol, 3.0 equiv) in toluene (6.0 mL) at0° C. The mixture was stirred at 0° C. for 90 min whereupon a solutionof 2-nitrobenzenesulfonyl hydrazine (651 mg, 3.00 mmol, 3.0 equiv) intetrahydrofuran (3 mL) was added dropwise via cannula. The resultingmixture was stirred at 0° C. for 10 min, then was allowed to warm to 23°C.; stirring was continued at that temperature for 23 h. An aqueouspotassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) was added andthe resulting biphasic mixture was extracted with dichloromethane (2×50mL). The organic extracts were combined and dried over anhydrous sodiumsulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a yellow sludge. The product was purified byflash column chromatography (95:5 to 1:9 ethyl acetate-hexanes),affording the alkene DRS3 as a white solid (356 mg, 74%).

R_(f) 0.65 (1:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.46(d, 2H, J=6.8 Hz, ArH), 7.39-7.34 (m, 3H, ArH), 5.81 (m, 1H, ═CHCH₂),5.55 (dd, 1H, J=10.3, 2.0 Hz, ═CHCOSi), 5.39 (d, 1H, J=12.2 Hz,OCHH′Ph), 5.35 (d, 1H, J=12.2 Hz, OCHH′Ph), 4.15 (s, 1H, CHOSi), 4.04(bs, 1H, OH), 3.76 (d, 1H, J=10.7 Hz, CHN(CH₃)₂), 2.58 (dd, 1H, J=10.7,3.9 Hz, C₃CH), 2.47 (m, 8H, N(CH₃)₂, ═CCH₂), 0.86 (s, 9H, C(CH₃)₃),−0.05 (s, 3H, SiCH₃), −0.13 (s, 3H, SiCH₃); ¹³C NMR (125 MHz, CDCl₃) δ191.5, 183.3, 167.9, 135.3, 128.8, 128.7, 128.5, 127.4, 106.8, 78.3,72.6, 72.0, 67.9, 60.7, 43.0, 42.1, 26.0, 25.8, 23.6, 18.2, −4.6, −5.0;FTIR (neat), cm⁻¹ 3528 (w, OH), 2933 (s, CH), 1702 (s, C═O), 1600 (m),1507 (s), 1092 (s, C—O), 1061 (s, C—O); HRMS (ES) m/z calcd for(C₂₆H₃₆N₂O₅Si+H)⁺ 485.2472. found 485.2457.

Diol DRS4:

Acetic acid (83.0 μL, 1.44 mmol, 2.0 equiv) and a solution oftetrabutylammonium fluoride in tetrahydrofuran (1.0 M, 1.44 mL, 1.44mmol, 2.0 equiv) were added in sequence to a solution of the olefin DRS3(350 mg, 0.723 mmol, 1.0 equiv) in tetrahydrofuran (7.0 mL) at 0° C. Theresulting light gray solution was stirred at 0° C. for 30 min, then wasallowed to warm to 23° C.; stirring was continued at that temperaturefor 5 h. The reaction mixture was concentrated, providing a brown oil.The product was purified by flash column chromatography (1:4 to 1:1ethyl acetate-hexanes), affording the diol DRS4 as a waxy white solid(202 mg, 76%).

R_(f) 0.38 (1:1 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ7.51-7.48 (m, 2H, ArH), 7.42-7.36 (m, 3H, ArH), 5.84 (m, 1H, ═CHCH₂),5.55 (m, 1H, ═CHCOH), 5.36 (m, 2H, OCH₂Ph), 4.15 (d, 1H, J=8.1 Hz,CHOH), 3.69 (d, 1H, J=8.8 Hz, CHN(CH₃)₂), 2.67 (m, 1H, C₃CH), 2.47 (s,6H, N(CH₃)₂), 2.43 (dd, 1H, J=7.7, 1.5 Hz, ═CCHH′), 2.36 (m, 1H,═CCHH′); FTIR (neat), cm⁻¹ 3492 (w, OH), 3272 (s, OH), 1703 (s, C═O),1606 (m), 1509 (s), 1008 (s, C—O), 732 (s); HRMS (ES) m/z calcd for(C₂₀H₂₂N₂O₅+H)⁺ 371.1607. found 371.1601.

Cyclohexenone DRS5:

Solid o-iodoxybenzoic acid (558 mg, 1.99 mmol, 3.0 equiv) was added to asolution of the diol DRS4 (246 mg, 0.665 mmol, 1.0 equiv) indimethylsulfoxide (5.0 mL) at 23° C. The resulting heterogeneous mixturewas stirred for 5 min whereupon it became homogeneous. The brownreaction mixture was stirred at 23° C. for 36 h. Water (10 mL) was addedresulting in the precipitation of excess o-iodoxybenzoic acid. Themixture was filtered and the filtrate was partitioned between saturatedaqueous sodium bicarbonate solution-brine (1:1, 20 mL) and ethylacetate-hexanes (2:1, 45 mL). The organic phase was separated and theaqueous phase was further extracted with a 45-mL portion of ethylacetate-hexanes (2:1). The organic extracts were combined and washedwith aqueous sodium sulfite solution (2.0 M, 50 mL), brine (50 mL), anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing the cyclohexenone DRS5 as alight brown foam (206 mg, 84%).

R_(f) 0.15 (1:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.48(d, 2H, J=7.3 Hz, ArH), 7.40-7.34 (m, 3H, ArH), 6.98 (m, 1H, ═CHCH₂),6.12 (ddd, 1H, J=12.2, 2.0, 2.0 Hz, ═CHC(O)), 5.35 (m, 2H, OCH₂Ar), 4.75(bs, 1H, OH), 3.85 (d, 1H, J=9.8 Hz, CHN(CH₃)₂), 2.82 (m, 3H, C₃CH,═CCH₂), 2.48 (s, 6H, N(CH₃)₂); ¹³C NMR (125 MHz, CDCl₃) δ 192.8, 188.2,182.8, 167.6, 149.7, 135.0, 128.9, 128.8, 128.6, 128.3, 107.9, 79.7,72.8, 60.4, 45.5, 42.4, 25.4; FTIR (neat), cm⁻¹ 3447 (w, OH), 1707 (s,C═O), 1673 (s, C═O), 1600 (m), 1512 (s), 1018 (s, C—O), 730 (s); HRMS(ES) m/z calcd for (C₂₀H₂₀N₂O₅+H)⁺ 369.1450. found 369.1454.

Silyl-Cyclohexenone DRS6:

2,6-Lutidine (75.0 μL, 0.640 mmol, 5.0 equiv) andtert-butyldimethylsilyl trifluoromethanesulfonate (88.0 μL, 0.380 mmol,3.0 equiv) were added in sequence to a solution of the cyclohexenoneDRS5 (47.0 mg, 0.130 mmol, 1.0 equiv) in dichloromethane (3 mL) at 23°C. The mixture was stirred at 23° C. for 3 h, then an aqueous potassiumphosphate buffer solution (pH 7.0, 0.2 M, 15 mL) was added. The biphasicmixture was extracted with dichloromethane (2×20 mL) and the organicextracts were combined and dried over anhydrous sodium sulfate. Thedried solution was filtered and the filtrate was concentrated, affordingthe silyl-cyclohexenone DRS6 as a white crystalline solid (56.0 mg,91%).

Mp 157-158° C. (dec); R_(f) 0.54 (1:3 ethyl acetate-hexanes); ¹H NMR(500 MHz, CDCl₃) δ 7.51 (d, 2H, J=1.5 Hz, ArH), 7.50-7.34 (m, 3H, ArH),6.94 (m, 1H, ═CHCH₂), 6.10 (ddd, 1H, J=10.3, 1.5, 1.5 Hz, ═CHC(O)), 5.36(m, 2H, OCH₂Ar), 3.79 (d, 1H, J=10.7 Hz, CHN(CH₃)₂), 2.83 (m, 2H,═CCH₂), 2.78 (m, 1H, C₃CH), 2.46 (s, 6H, N(CH₃)₂), 0.84 (s, 9H,C(CH₃)₃), 0.27 (s, 3H, SiCH₃), 0.06 (s, 3H, SiCH₃); ¹³C NMR (125 MHz,CDCl₃) δ 193.4, 187.9, 181.6, 167.7, 149.5, 135.2, 128.8, 128.8, 128.8,128.6, 108.6, 83.5, 72.8, 59.8, 48.1, 42.2, 26.3, 25.8, 19.3, −2.2,−3.8; FTIR (neat), cm⁻¹ 2942 (s), 1719 (s, C═O), 1678 (s, C═O), 1602(m), 1510 (s), 1053 (s, C—O), 733 (s); HRMS (ES) m/z calcd for(C₂₆H₃₄N₂O₅Si+H)⁺ 483.2315. found 483.2321.

Ketone MGC9:

A solution of methylmagnesium bromide in ether (3.15 M, 11.6 mL, 36.7mmol, 1.07 equiv) was added to a solution of the aldehyde MGC8(synthesized in 2 steps from commercially available 3-benzyloxy benzylalcohol as reported by: Hollinshed, S. P.; Nichols, J. B.; Wilson, J. W.J. Org. Chem. 1994, 59, 6703.) (10.0 g, 34.3 mmol, 1.0 equiv) intetrahydrofuran (90 mL) at −5° C. (NaCl/ice bath). The light brownsolution was stirred at −5° C. for 60 min, then was partitioned betweensaturated aqueous ammonium chloride solution (400 mL) and ethyl acetate(400 mL). The organic phase was separated and dried over anhydroussodium sulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a light yellow oil (10.1 g, 95% crude). Theproduct was used without further purification.

Sodium bromide (846 mg, 8.22 mmol, 0.25 equiv) and2,2,6,6-tetramethyl-1-piperidinyloxyl (51.0 mg, 0.329 mmol, 0.01 equiv)were added in sequence to a solution of the light yellow oil preparedabove (10.1 g, 32.8 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) at 0° C.A freshly prepared solution of sodium bicarbonate (690 mg, 8.22 mmol,0.25 equiv) in commercial Clorox bleach (90 mL) was cooled to 0° C. andwas added in one portion to the mixture prepared above at 0° C. Theresulting bright yellow mixture was stirred vigorously at 0° C. for 1.5h whereupon sodium sulfite (1.0 g) was added. The resulting mixture wasstirred for 15 min at 23° C., then was partitioned between water (400mL) and ethyl acetate (400 mL). The organic phase was separated anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a light brown oil. The productwas crystallized from ethanol, furnishing the ketone MGC9 as a whitesolid (8.08 g, 80% over 2 steps).

R_(f) 0.80 (3:7 ethyl acetate-hexanes); ¹H NMR (400 MHz, CDCl₃) δ7.26-7.48 (m, 6H, ArH), 6.98 (m, 2H, ArH), 5.19 (s, 2H, OCH₂Ph), 2.62(s, 3H, C(═O)CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 202.4, 155.5, 144.4,136.3, 128.9, 128.7, 128.3, 127.2, 120.3, 115.2, 109.1, 71.3, 30.9; FTIR(neat), cm⁻¹ 3065 (w), 3032 (w), 2918 (m), 1701 (s, C═O), 1565 (m), 1426(m), 1300 (s), 1271 (s), 1028 (m); HRMS (ES) m/z calcd for(C₁₅H₁₃O₂Br+H)⁺ 304.0099. found 304.0105.

Epoxide MGC10:

Dimethylsulfoxide (90 mL) was added dropwise via syringe to a mixture ofsolid trimethylsulfoxonium iodide (694 mg, 3.15 mmol, 1.3 equiv) andsolid sodium hydride (60% in oil, 126 mg, 3.15 mmol, 1.3 equiv, washedwith three 2-mL portions of n-hexane) at 23° C. Vigorous gas evolutionwas observed upon addition. The resulting cloudy gray mixture wasstirred at 23° C. for 40 min, then a solution of the ketone MGC9 (8.08g, 26.5 mmol, 1.0 equiv) in dimethylsulfoxide (30 mL) was added dropwisevia cannula. The transfer was quantitated with a 2-mL portion ofdimethylsulfoxide. The resulting orange mixture was stirred at 23° C.for 35 h, then was partitioned between brine (1 L) and ether (500 mL).The organic phase was separated and the aqueous phase was furtherextracted with one 500-mL portion of ether. The organic phases werecombined and dried over anhydrous sodium sulfate. The dried solution wasfiltered and the filtrate was concentrated, providing a yellow oil. Theproduct was purified by flash column chromatography (5:95 ethylacetate-hexanes), affording the epoxide MGC10 as a clear oil (7.94 g,94%).

R_(f) 0.90 (3:7 ethyl acetate-hexanes); ¹H NMR (300 MHz, CDCl₃) δ7.20-7.52 (m, 6H, ArH), 7.10 (dd, 1H, J=7.5, 1.2 Hz, o-ArH), 6.88 (dd,1H, J=8.1, 1.2 Hz, o-ArH), 5.16 (s, 2H, OCH₂Ph), 3.03 (d, 1H, J=4.8 Hz,CHH′OCCH₃), 2.87 (d, 1H, J=4.8 Hz, CHH′OCCH₃), 1.67 (s, 3H, COCH₃); ¹³CNMR (100 MHz, CDCl₃) δ 155.0, 143.4, 136.7, 128.8, 128.4, 128.2, 127.2,121.2, 112.8, 112.3, 71.2, 59.7, 55.9, 22.9; FTIR (neat), cm⁻¹ 3034 (w),2981 (w), 2925 (w), 1595 (w), 1567 (s), 1469 (s), 1423 (s), 1354 (s),1300 (s), 1266 (s), 1028 (s); HRMS (ES) m/z calcd for (C₁₆H₁₅O₂Br+H)⁺318.0255. found 318.0254.

Benzocyclobutenol MGC11:

A solution of n-butyllithium in hexanes (1.60 M, 8.25 mL, 13.6 mmol, 1.4equiv) was added dropwise via syringe down the side of a reaction vesselcontaining a solution of the epoxide MGC10 (3.11 g, 9.74 mmol, 1.0equiv) in tetrahydrofuran (90 mL) at −78° C. The resulting yellowsolution was stirred at −78° C. for 20 min whereupon a suspension ofmagnesium bromide (3.95 g, 21.4 mmol, 2.2 equiv) in tetrahydrofuran (25mL) was added dropwise via cannula. The transfer was quantitated withtwo 2.5-mL portions of tetrahydrofuran. The resulting cloudy mixture wasstirred at −78° C. for 60 min, then the cooling bath was removed and thereaction mixture was allowed to warm to 23° C. The mixture became clearupon warming and was stirred at 23° C. for 1 h. The reaction mixture waspoured into aqueous Rochelle's salt solution (10% wt/wt, 1 L) and theresulting mixture was extracted with ethyl acetate (2×400 mL). Theorganic phases were combined and dried over anhydrous sodium sulfate.The dried solution was filtered and the filtrate was concentrated,providing an off-white solid. The product was purified by flash columnchromatography (1:9 to 2:9 ethyl acetate-hexanes), affording thetrans-benzocyclobutenol MGC11 as a white solid (1.57 g, 67%).

R_(f) 0.50 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.44(br d, 2H, J=7.5 Hz, ArH), 7.38 (br t, 2H, J=7.5 Hz, ArH), 7.22-7.34 (m,2H, ArH), 6.82 (d, 1H, J=8.5 Hz, o-ArH), 6.75 (d, 1H, J=7.5 Hz, o-ArH),5.35 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.25 (d, 1H, J=12.0 Hz, OCHH′Ph),),4.71 (br d, 1H, J=5.5 Hz, CHOH), 3.31 (br q, 1H, J=7.0 Hz, CHCH₃), 2.21(br d, 1H, J=7.0 Hz, OH), 1.38 (d, 3H, J=7.0 Hz, CHCH₃); ¹³C NMR (100MHz, CDCl₃) δ 154.0, 148.9, 137.4, 131.5, 128.5, 128.4, 127.8, 127.3,115.2, 114.6, 77.6, 71.2, 50.6, 16.5; FTIR (neat), cm⁻¹ 3249 (m, OH),2958 (w), 1602 (m), 1580 (s), 1453 (s), 1261 (s), 1039 (s); HRMS (ES)m/z calcd for (C₁₆H₁₆O₂+H)⁺ 240.1150. found 240.1154.

Benzocyclobutenol MGC12:

Triethylamine (336 μL, 2.41 mmol, 1.4 equiv) and triethylsilyltrifluoromethanesulfonate (468 μL, 2.07 mmol, 1.2 equiv) were added insequence to a solution of the benzocyclobutenol MGC11 (500 mg, 1.72mmol, 1.0 equiv) in dichloromethane (10 mL) at 23° C. The light yellowsolution was stirred at 23° C. for 15 min, then was partitioned betweenwater (30 mL) and dichloromethane (30 mL). The organic phase wasseparated and dried over anhydrous sodium sulfate. The dried solutionwas filtered and the filtrate was concentrated, providing a yellow oil.The product was purified by flash column chromatography (5:95 ethylacetate-hexanes), affording the benzocyclobutenol MGC12 (609 mg, 99%) asa clear oil.

R_(f) 0.85 (1:4 ethyl acetate-hexanes); ¹H NMR (400 MHz, CDCl₃) δ7.48-7.32 (m, 5H, ArH), 7.24 (m, 2H, ArH), 6.82 (d, 1H, J=8.4 Hz,o-ArH), 6.74 (d, 1H, J=7.2 Hz, o-ArH), 5.37 (d, 1H, J=11.2 Hz,CHH′Ph),), 5.20 (d, 1H, J=11.2 Hz, CHH′Ph),), 4.87 (d, 1H, J=1.6 Hz,CHOTES), 3.45 (dq, 1H, J=7.2, 1.6 Hz, CHCH₃), 1.42 (d, 3H, J=7.2 Hz,CHCH₃), 0.98 (t, 9H, J=7.6 Hz, TES), 0.56 (q, 6H, J=7.6 Hz, TES); ¹³CNMR (100 MHz, CDCl₃) δ 154.2, 148.8, 137.6, 131.3, 129.0, 128.7, 128.1,127.8, 115.1, 114.7, 71.7, 49.9, 16.9, 7.1, 5.2, 5.1; FTIR (neat), cm⁻¹2952 (w), 2923 (w), 2854 (w), 1606 (w), 1469 (w), 1371 (m), 1265 (s),1086 (s), 1057 (s), 1048 (s); HRMS (ES) m/z calcd for (C₂₂H₃₀O₂Si+H)⁺354.2015. found 354.2006.

Vinyl Sulfide MGC13:

Solid pyridinium hydrobromide perbromide (293 mg, 0.917 mmol, 2.5 equiv)was added to a solution of the cyclohexenone DRS5 (135 mg, 0.367 mmol,1.0 equiv) in dichloromethane (4 mL) at 23° C. The brown solution wasstirred vigorously at 23° C. for 17 h whereupon sodium sulfite (150 mg,1.19 mmol, 3.25 equiv) was added. The resulting mixture was partitionedbetween an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M,30 mL) and dichloromethane (30 mL). The organic phase was separated anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a light brown foamy solid. Theproduct was used immediately without further purification. R_(f) 0.45(2:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.24 (d, 2H, J=7.0Hz, o-ArH), 7.02 (t, 2H, J=7.0 Hz, m-ArH), 6.99 (d, 1H, J=7.0 Hz,p-ArH), 6.42 (ddd, 1H, J=6.0, 3.5, 2.0 Hz, CH═CBr), 5.12 (d, 1H, J=12.5Hz, CHH′Ph),), 5.03 (d, 1H, J=12.5 Hz, CHH′Ph),), 4.00 (br s, 1H, OH),3.25 (d, 1H, J=11.0 Hz, CHN(CH₃)₂), 2.28-2.22 (m, 2H, CH₂CH, CH₂CH),2.16 (dd, 1H, J=18.0, 6.0 Hz, CH₂CH), 1.83 (s, 6H, N(CH₃)₂); FTIR(neat), cm⁻¹ 3397 (m, OH), 3063 (m), 2943 (m), 1714 (s, C═O), 1606 (s),1514 (s), 1477 (s), 1371 (m), 1022 (m); HRMS (ES) m/z calcd for(C₂₀H₁₉O₅BrN₂)⁺ 447.0555. found 447.0545.

Benzenethiol (39.0 μL, 0.378 mmol, 1.03 equiv) and1,8-diazabicyclo[5,4,0]undec-7-ene (56.0 μL, 0.378 mmol, 1.03 equiv)were added in sequence to a solution of the product prepared above (164mg, 0.367 mmol, 1.0 equiv) in N,N-dimethylformamide (4 mL) at 0° C. Theresulting dark brown mixture was stirred vigorously at 0° C. for 25 min,then was partitioned between ethyl acetate-hexanes (1:1, 30 mL) and anaqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL). Theorganic phase was separated and the aqueous phase was further extractedwith two 15-mL portions of ethyl acetate-hexanes (1:1). The organicphases were combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing abrown oil. The product was purified by flash column chromatography(15:85 to 1:4 ethyl acetate-hexanes), furnishing the vinyl sulfide MGC13as a white foam (116 mg, 66% over two steps).

R_(f) 0.47 (2:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.34(dd, 2H, J=7.0, 1.0 Hz, o-ArH), 7.23 (d, 2H, J=6.5 Hz, o-ArH), 6.85-7.04(m, 6H, ArH), 6.27 (ddd, 1H, J=6.0, 3.0, 1.0 Hz, CH═CSPh), 5.11 (d, 1H,J=12.0 Hz, OCHH′Ph), 5.02 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.62 (br s, 1H,OH), 3.42 (d, 1H, J=10.5 Hz, CHN(CH₃)₂), 2.44 (ddd, 1H, J=20.0, 5.5, 3.0Hz, CH₂CH), 2.27-2.34 (m, 2H, CH₂CH, CH₂CH), 1.87 (s, 6H, N(CH₃)₂); ¹³CNMR (100 MHz, CDCl₃) δ 188.9, 187.4, 182.5, 167.6, 145.4, 135.3, 135.2,132.8, 132.6, 129.5, 128.6, 128.4, 128.3, 128.0, 127.8, 108.1, 80.3,72.5, 59.8, 45.7, 41.4, 25.9; FTIR (neat), cm⁻¹ 3445 (w, OH), 3056 (w),2943 (m), 2800 (w), 1711 (s, C═O), 1682 (s), 1600 (m), 1507 (s), 1471(s), 1451 (m), 1333 (m), 1020 (m); HRMS (ES) m/z calcd for(C₂₆H₂₄O₅N₂S+H)⁺ 477.1484. found 447.1465.

Diel-Alder Addition Product MGC14 and Lactone MGC15:

A reaction vessel containing a mixture of the vinylsulfide MGC13 (131mg, 0.275 mmol, 1.0 equiv) and the benzocyclobutenol MGC12 (750 mg, 2.11mmol, 7.7 equiv) was placed in an oil bath preheated to 85° C. The lightyellow solution was stirred at 85° C. for 48 h, then was allowed to coolto 23° C. The cooled mixture was purified by flash column chromatography(1:19 to 1:4 ethyl acetate-hexanes), affording the Diels-Alder additionproduct MGC14 as an off-white foamy solid (145 mg, 64%), the lactoneMGC15 as a clear oil (20.0 mg, 9%), and the recovered benzocyclobutenolMGC12 as a clear oil (650 mg).

Diels-Alder Addition Product MGC14:

mp 178-179° C.; R_(f) 0.55 (2:3 ethyl acetate-hexanes); ¹H NMR (600 MHz,C₆D₆) δ 7.27 (d, 2H, J=7.2 Hz, o-ArH), 7.06-7.22 (m, 8H, ArH), 6.92-6.96(m, 3H, ArH), 6.85 (d, 1H, J=7.2 Hz, ArH), 6.70-6.75 (m, 3H, ArH), 6.55(d, 1H, J=8.4 Hz, o-ArH), 5.75 (s, 1H, CHOTES), 5.29 (br s, 1H, OH),5.16 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.10 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.66(d, 1H, J=10.8 Hz, OCHH′Ph′), 4.63 (d, 1H, J=10.8 Hz, OCHH′Ph′), 4.36(d, 1H, J=6.6 Hz, CHN(CH₃)₂), 3.02 (dq, 1H, J=7.8, 6.0 Hz, CH₃CH), 2.77(ddd, 1H, J=6.6, 6.0, 4.2 Hz, CHCHN(CH₃)₂), 2.41-2.52 (m, 2H, CHCHH′CH,CH₃CHCHCH₂), 2.08 (s, 6H, N(CH₃)₂), 1.83 (ddd, 1H, J=13.2, 4.2, 4.2 Hz,CHCHH′CH), 1.34 (d, 3H, J=7.8 Hz, CH₃CH), 0.70 (t, 9H, J=7.8 Hz,Si(CH₂CH₃)₃), 0.48 (d, 6H, J=7.8 Hz, Si(CH₂CH₃)₃); ¹³C NMR (100 MHz,CDCl₃) δ 196.3, 186.1, 181.4, 168.3, 156.3, 143.9, 137.6, 136.6, 135.4,130.6, 129.8, 129.3, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8, 125.4,121.1, 109.3, 108.4, 80.6, 72.4, 70.2, 66.0, 62.5, 61.7, 43.2, 42.0,38.1, 37.2, 27.4, 20.5, 6.9, 4.9; FTIR (neat), cm⁻¹ 3490 (w, OH), 3063(w), 3023 (w), 2951 (m), 2871 (m), 1715 (s, C═O), 1602 (m), 1589 (m),1513 (s), 1457 (s), 1366 (m), 1260 (s), 1065 (s), 1012 (s); HRMS (FAB)m/z calcd for (C₄₈H₅₄O₇N₂SSi+Na)⁺ 853.3318. found 853.3314.

Lactone MGC15:

R_(f) 0.55 (3:7 ethyl acetate-hexanes); ¹H NMR (600 MHz, C₆D₆) δ 7.34(d, 2H, J=7.2 Hz, o-ArH), 7.02-7.18 (m, 11H, ArH), 6.72-6.84 (m, 4H,ArH), 6.54 (d, 1H, J=7.8 Hz, o-ArH), 5.73 (s, 1H, CHOTES), 5.49 (d, 1H,J=6.6 Hz, (C═O)OCHC═O), 5.20 (s, 2H, OCH₂Ph), 4.60 (d, 1H, J=11.4 Hz,OCHH′Ph′), 4.57 (d, 1H, J=11.4 Hz, OCHH′Ph′), 3.49 (d, 1H, J=11.4 Hz,CHN(CH₃)₂), 3.23 (dq, 1H, J=9.0, 7.2 Hz, CH₃CH), 2.49 (m, 1H,CH₃CHCHCHH′), 2.30-2.40 (m, 2H, CHCHN(CH₃)₂, CH₃CHCHCH₂), 2.16 (dd, 1H,J=12.0, 0.6 Hz, CH₃CHCHCHH′), 1.96 (s, 6H, N(CH₃)₂), 1.33 (d, 3H, J=7.2Hz, CH₃CH), 0.73 (t, 9H, J=7.8 Hz, Si(CH₂CH₃)₃), 0.46-0.62 (m, 6H,Si(CH₂CH₃)₃); ¹³C NMR (100 MHz, CDCl₃) δ 196.4, 176.0, 170.0, 157.9,156.0, 144.0, 136.6, 136.5, 135.6, 129.8, 129.7, 129.4, 128.9, 128.6,128.4, 128.3, 128.2, 128.1, 127.8, 125.1, 121.2, 108.8, 101.9, 75.9,72.1, 70.1, 64.7, 64.6, 62.9, 41.4, 36.7, 35.6, 27.7, 21.7, 6.9, 4.9;FTIR (neat), cm⁻¹ 3062 (w), 3033 (w), 2950 (m), 2874 (m), 1731 (s, C═O),1599 (m), 1590 (m), 1514 (s), 1453 (s), 1365 (m), 1259 (s), 1120 (s),1059 (s), 1010 (s); HRMS (ES) m/z calcd for (C₄₈H₅₄O₇N₂SSi+H)⁺ 831.3499.found 831.3509.

Alcohol MGC16:

Triethylamine trihydrofluoride (200 μL, 1.23 mmol, 8.5 equiv) was addedto a solution of the Diels-Alder addition product MGC14 (120 mg, 0.144mmol, 1.0 equiv) in tetrahydrofuran (6 mL) at 23° C. The mixture wasstirred vigorously at 23° C. for 12 h, then was partitioned between anaqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) andethyl acetate (30 mL). The organic phase was separated and dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a light brown solid. The productwas purified by flash column chromatography (1:4 to 1:1 ethylacetate-hexanes), affording the alcohol MGC16 as a colorless oil (78.3mg, 76%).

R_(f) 0.20 (2:3 ethyl acetate-hexanes); ¹H NMR (600 MHz, C₆D₆) δ 7.69(dd, 2H, J=7.2, 0.6 Hz, o-ArH), 7.24 (d, 2H, J=7.2 Hz, ArH), 6.92-7.06(m, 12H, ArH), 6.76 (d, 1H, J=7.8 Hz, ArH), 6.47 (d, 1H, J=8.4 Hz,o-ArH), 5.44 (br s, 1H, CHOH), 5.18 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.16(d, 1H, J=12.0 Hz, OCHH′Ph), 4.57 (d, 1H, J=12.6 Hz, OCHH′Ph′), 4.52 (d,1H, J=12.6 Hz, OCHH′Ph′), 3.44 (dq, 1H, J=6.6, 5.4 Hz, CH₃CH), 2.98 (d,1H, J=3.0 Hz, CHN(CH₃)₂), 2.90 (m, 1H, CHCHN(CH₃)₂), 2.76 (br s, 1H,OH), 2.32 (m, 1H, CH₃CHCHCH₂), 1.94 (m, 1H, CH₃CHCHCH₂), 1.79 (s, 6H,N(CH₃)₂), 1.07 (m, 1H, CH₃CHCHCH₂), 0.84 (d, 3H, J=6.6 Hz, CH₃CH); ¹³CNMR (100 MHz, CDCl₃) δ 202.5, 185.6, 179.2, 168.9, 156.9, 139.4, 139.1,137.1, 136.5, 135.3, 130.5, 129.6, 128.8, 128.7, 128.6, 128.5, 128.4,128.3, 127.8, 126.9, 124.7, 119.3, 110.0, 106.8, 82.3, 72.5, 69.9, 66.4,64.2, 59.3, 43.0, 39.1, 37.8, 32.6, 25.3, 16.8; FTIR (neat), cm⁻¹ 3435(w, OH), 3066 (w), 2964 (w), 2933 (w), 2871 (w), 1738 (s, C═O), 1698 (s,C═O), 1614 (m), 1583 (m), 1513 (s), 1471 (s), 1453 (s), 1369 (m), 1263(m), 1035 (m), 1014 (m); HRMS (ES) m/z calcd for (C₄₂H₄₀O₇N₂S+H)⁺717.2634. found 717.2631.

Triketone MGC17:

Solid o-iodoxybenzoic acid (459 mg, 1.64 mmol, 15.0 equiv) was added inone portion to a solution of the alcohol MGC16 (78.3 mg, 0.109 mmol, 1.0equiv) in dimethylsulfoxide (3.0 mL) at 23° C. The resultingheterogeneous mixture was stirred for 5 min whereupon it becamehomogeneous. The reaction vessel was protected from light and was placedin an oil bath preheated to 35° C. The brown solution was stirredvigorously at 35° C. for 18 h, then was partitioned between saturatedaqueous sodium bicarbonate solution-brine-water (2:1:1, 75 mL) and ethylacetate-ether (1:2, 35 mL). The organic phase was separated and theaqueous phase was further extracted with two 25-mL portions ethylacetate-ether (1:2). The organic phases were combined and dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a yellow oil. The product waspurified by flash column chromatography (1:2 ethyl acetate-hexanes),affording the ketone MGC17 as a yellow oil (61.7 mg, 79%).

R_(f) 0.45 (2:3 ethyl acetate-hexanes); ¹H NMR (600 MHz, C₆D₆) δ 7.57(d, 2H, J=7.2 Hz, o-ArH), 7.40 (d, 2H, J=7.2 Hz, ArH), 7.18-7.23 (m, 3H,ArH), 6.94-7.06 (m, 6H, ArH), 6.76-6.84 (m, 3H, ArH), 6.59 (d, 1H, J=7.8Hz, ArH), 6.53 (d, 1H, J=8.4 Hz, o-ArH), 5.09 (d, 1H, J=12.6 Hz,OCHH′Ph), 4.96 (d, 1H, J=12.6 Hz, OCHH′Ph), 4.77 (d, 1H, J=12.0 Hz,OCHH′Ph′), 4.72 (d, 1H, J=12.0 Hz, OCHH′Ph′), 4.48 (br s, 1H, OH), 4.06(dq, 1H, J=7.2, 3.0 Hz, CH₃CH), 3.15 (d, 1H, J=12.0 Hz, CHN(CH₃)₂), 2.20(ddd, 1H, J=12.6, 5.4, 3.0 Hz, CH₃CHCHCH₂), 2.13 (ddd, 1H, J=12.0, 3.0,0.6 Hz, CHCHN(CH₃)₂), 1.81-1.88 (m, 7H, N(CH₃)₂, CH₃CHCHCHH′), 1.78(ddd, 1H, J=13.8, 5.4, 0.6 Hz, CH₃CHCHCHH′), 1.01 (d, 3H, J=7.2 Hz,CH₃CH); ¹³C NMR (100 MHz, CDCl₃) δ 200.3, 187.5, 183.1, 167.8, 160.6,146.4, 138.2, 137.1, 135.3, 134.3, 131.7, 129.6, 128.9, 128.6, 128.5,128.4, 128.3, 127.7, 126.7, 121.3, 118.0, 112.8, 108.3, 82.9, 77.5,72.4, 70.3, 58.1, 47.0, 44.1, 32.4, 18.7, 18.0, 16.3; FTIR (neat), cm⁻¹3457 (w, OH), 3063 (w), 2939 (w), 2878 (w), 2795 (w), 1727 (s, C═O),1704 (s, C═O), 1667 (m, C═O), 1593 (s), 1513 (s), 1471 (s), 1453 (s),1371 (m), 1276 (m), 1044 (m); HRMS (ES) m/z calcd for (C₄₂H₃₈O₇N₂S+H)⁺715.2478. found 715.2483.

Peroxide MGC18:

A solution of trifluoroacetic acid in dichloromethane (1.0 M, 0.189 mL,0.189 mmol, 2.5 equiv) and a solution of m-chloroperoxybenzoic acid indichloromethane (0.5 M, 0.228 mL, 0.114 mmol, 1.5 equiv) were added insequence to a solution of the sulfide MGC17 (54.2 mg, 0.0758 mmol, 1.0equiv) in dichloromethane (4.0 mL) at −78° C. The resulting cloudymixture was stirred at −78° C. for 10 min, then the −78° C. bath wasreplaced with a 0° C. bath. The mixture became homogeneous upon warming.The solution was stirred at 0° C. for 30 min, then was partitionedbetween an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M,10 mL) and dichloromethane (10 mL). The organic phase was separated anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a bright yellow oil. The oilwas taken up in toluene (1 mL) and dried by azeotropic distillation at40° C. under high vacuum. The resulting yellow oil was dissolved inchloroform (2 mL) and the reaction vessel was exposed to atmosphericoxygen. The mixture was allowed to stand until oxidation was complete asevidenced by ¹H NMR spectroscopy. The mixture was filtered and thefiltrate was concentrated, providing the peroxide MGC18 as a brown oil.The product was reduced immediately to tetracycline.

The peroxide MGC18 can also be prepared by following the procedurereported by Wasserman (J. Am. Chem. Soc. 1986, 108, 4237-4238.):

A solution of trifluoroacetic acid in dichloromethane (1.0 M, 24.5 μL,0.0245 mmol, 2.5 equiv) and a solution of m-chloroperoxybenzoic acid indichloromethane (0.5 M, 29.4 μL, 0.0147 mmol, 1.5 equiv) were added insequence to a solution of the sulfide MGC17 (7.00 mg, 0.00979 mmol, 1.0equiv) in dichloromethane (0.5 mL) at −78° C. The resulting cloudymixture was stirred at −78° C. for 10 min, then the −78° C. bath wasreplaced with a 0° C. bath. The mixture became homogeneous upon warming.The solution was stirred at 0° C. for 30 min, then was partitionedbetween an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 8mL) and dichloromethane (8 mL). The organic phase was separated anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a bright yellow oil. The oilwas taken up in toluene (1 mL) and dried by azeotropic distillation at40° C. under high vacuum. The resulting yellow oil was dissolved inchloroform (2 mL) and meso-tetraphenylporphine (0.6 mg, 0.979 μmol, 0.10equiv) was added in one portion. Oxygen gas was bubbled through theresulting mixture under UV irradiation (200 W Hg lamp) for 10 min. Themixture was concentrated to 0.5 mL and was diluted with methanol (5 mL)resulting in precipitation of meso-tetraphenylporphine. The resultingmixture was filtered and the filtrate was concentrated, providing theperoxide MGC18 a light yellow solid.

R_(f) 0.10 (2:3 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆, ketotautomer reported) δ 8.95 (br s, 1H, OOH), 7.48 (d, 2H, J=7.0 Hz,o-ArH), 7.28 (d, 2H, J=7.0 Hz, ArH), 6.96-7.16 (m, 8H, ArH), 6.53 (d,1H, J=8.0 Hz, ArH), 5.14 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.03 (d, 1H,J=12.0 Hz, OCHH′Ph), 4.83 (d, 1H, J=12.5 Hz, OCHH′Ph′), 4.74 (d, 1H,J=12.5 Hz, OCHH′Ph′), 4.60 (br s, 1H, OH), 3.54 (d, 1H, J=11.0 Hz,CHCHN(CH₃)₂), 3.12 (dd, 1H, J=18.0, 0.5 Hz, CHCHH′CH), 2.82 (dd, 1H,J=18.0, 4.5 Hz, CHCHH′CH), 2.44 (ddd, 1H, J=11.0, 4.5, 0.5 Hz,CHCHN(CH₃)₂), 1.86 (s, 6H, N(CH₃)₂), 1.01 (s, 3H, CH₃); ¹³C NMR (100MHz, C₆D₆, enol and keto tautomers reported) δ 194.4, 188.6, 187.8,187.2, 182.3, 178.4, 171.9, 167.7, 165.6, 159.5, 158.4, 147.9, 145.9,137.0, 136.8, 136.6, 135.4, 135.3, 134.5, 134.3, 133.5, 133.4, 133.1,132.9, 131.0, 130.8, 130.2, 129.9, 129.7, 129.2, 128.9, 126.8, 126.7,124.5, 124.3, 122.2, 118.6, 116.9, 116.5, 113.4, 113.3, 113.2, 108.2,107.9, 103.3, 83.7, 81.7, 80.1, 79.1, 72.4, 70.7, 70.4, 63.9, 59.1,46.1, 44.9, 41.4, 40.8, 31.5, 30.0, 26.8, 22.9, 21.4; FTIR (neat film),cm⁻¹ 3035 (w), 2946 (w), 1907 (w), 1731 (s, C═O), 1410 (s), 1379 (m),1235 (m), 1170 (m), 1136 (m); HRMS (ES) m/z calcd for (C₃₆H₃₂O₉N₂+H)⁺637.2186. found 637.2190.

(−)-Tetracycline (MGC29):

Pd black (14.1 mg, 0.133 mmol, 1.75 equiv) was added in one portion to asolution of the peroxide MGC18 (48.2 mg, 0.0758 mmol, 1.0 equiv) indioxane (3 mL) at 23° C. An atmosphere of hydrogen was introduced bybriefly evacuating the flask, then flushing with pure hydrogen (1 atm).The Pd catalyst was initially present as a fine dispersion, butaggregated into clumps within 5 min. The yellow heterogeneous mixturewas stirred at 23° C. for 2 h, then was filtered through a plug ofcotton. The filtrate was concentrated, affording a yellow solid. Theproduct was purified by preparatory HPLC on a Phenomenex Polymerx DVBcolumn (10 μM, 250×10 mm, flow rate 4.0 mL/min, Solvent A:methanol-0.005 N aq. HCl (1:4), Solvent B: acetonitrile) using aninjection volume of solvent A (500 μL) containing oxalic acid (10 mg)and an isochratic elution of 5% B for 2 min, then a gradient elution of5-50% B for 20 min. The peak eluting at 11-16 min was collected andconcentrated, affording (−)-tetracycline hydrochloride as a yellowpowder (16.0 mg, 44% from triketone MGC17), which was identical withnatural (−)-tetracycline hydrochloride in all respects.

¹H NMR (600 MHz, CD₃OD, hydrochloride) δ 7.50 (dd, 1H, J=8.4, 7.8 Hz,ArH), 7.13 (d, 1H, J=7.8 Hz, ArH), 6.91 (d, 1H, J=8.4 Hz, ArH), 4.03 (s,1H, CHN(CH₃)₂), 2.96-3.04 (m, 7H, HOC(CH₃)CH, N(CH₃)₂), 2.91 (br dd, 1H,J=12.6, 2.4 Hz, (CH₃)₂NCHCH), 2.18 (ddd, 1H, J=12.6, 6.0, 2.4 Hz,CHCHH′CH), 1.90 (ddd, 1H, J=12.6, 12.6, 12.0 Hz, CHCHH′CH), 1.60 (s, 3H,CH₃); ¹³C NMR (100 MHz, CD₃OD) δ 195.4, 174.5, 163.8, 148.3, 137.8,118.7, 116.4, 116.0, 107.5, 96.5, 74.7, 71.2, 70.1, 43.5, 43.0, 35.9,27.8, 22.9; UV max (0.1 N HCl), nm 217, 269, 356; [α]_(D)=−251° (c=0.12in 0.1 M HCl); lit. (The Merck Index: An Encyclopedia of Chemicals,Drugs, and Biologicals, 12^(th) ed. Budavari, S.; O'Neal, M. J.; Smith,A.; Heckelman, P. E.; Kinneary, J. F., Eds.; Merck & Co.: WhitehouseStation, N J, 1996; entry 9337.) UV max (0.1 N HCl), nm 220, 268, 355;[α]_(D)=−257.9° (c=0.5 in 0.1 M HCl); HRMS (ES) m/z calcd for(C₂₂H₂₄O₈N₂+H)⁺ 445.1611. found 445.1608.

Example 2—Synthesis of (−)-Doxycycline

Allylic Bromide MGC19:

Triphenylphosphine (297 mg, 1.13 mmol, 3.5 equiv) and carbontetrabromide (376 mg, 1.13 mmol, 3.5 equiv) were added in sequence to asolution of the allylic alcohol MGC6 (162 mg, 0.324 mmol, 1.0 equiv) inacetonitrile (2.5 mL) at 0° C. The resulting brown suspension wasstirred at 0° C. for 10 min, then the cooling bath was removed. Themixture was allowed to warm to 23° C. and stirring was continued at thattemperature for 10 min. The mixture was partitioned between ethylacetate (50 mL) and saturated aqueous sodium bicarbonate solution (40mL). The organic phase was separated and the aqueous phase was furtherextracted with an additional 50 mL-portion of ethyl acetate. The organicphases were combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing abrown oily solid. The product was purified by flash columnchromatography (1:9 to 2:8 ethyl acetate-hexanes), yielding the allylicbromide MGC19 (164 mg, 90%) as a white solid.

R_(f) 0.30 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.30(d, 2H, J=7.0, o-ArH), 7.06 (dd, 2H, J=7.0, 6.0 Hz, m-ArH), 7.01 (d, 1H,J=6.0, p-ArH), 5.75 (dd, 1H, J=10.5, 2.5 Hz, ═CHCHBr), 5.71 (m, 1H,CH═CHCHBr), 5.17 (d, 1H, J=11.5 Hz, OCHH′Ph), 5.07 (d, 1H, J=11.5 Hz,OCHH′Ph), 4.69 (m, 1H, ═CHCHBr), 4.43 (br s, 1H, OH), 4.24 (d, 1H, J=7.0Hz, CHOTBS), 3.57 (d, 1H, J=10.0 Hz, CHN(CH₃)₂), 2.69 (ddd, 1H, J=10.0,4.5, 0.5 Hz, CHCHN(CH₃)₂), 1.92 (s, 6H, N(CH₃)₂), 0.99 (s, 9H,SiC(CH₃)₃), 0.22 (s, 3H, SiCH₃), −0.02 (s, 3H, SiCH₃); ¹³C NMR (125 MHz,C₆D₆) δ 189.3, 181.3, 167.8, 135.2, 129.5, 128.6, 128.6, 128.5, 128.2,127.6, 107.3, 80.8, 76.9, 72.4, 64.8, 54.6, 46.3, 41.5, 26.2, 18.4,−2.9, −4.2; FTIR (neat), cm⁻¹ 3499 (m, OH), 2930 (m), 2856 (m), 2799(w), 1704 (s, C═O), 1605 (s), 1514 (s), 1471 (s), 1362 (s), 1255 (s),1144 (s), 1053 (s); HRMS (ES) m/z calcd for (C₂₆H₃₅BrN₂O₅Si+H)⁺563.1577. found 563.1575.

Allylic Sulfide MGC20:

Triethylamine (0.229 mL, 1.64 mmol, 1.3 equiv) and benzenethiol (0.150mL, 1.45 mmol, 1.15 equiv) were added in sequence to a solution of theallylic bromide MGC19 (712 mg, 1.26 mmol, 1.0 equiv) in acetonitrile (17mL) at 0° C. The mixture was stirred at 0° C. for 20 min, then thecooling bath was removed. The reaction mixture was allowed to warm to23° C. and stirring was continued at that temperature for 10 min. Thereaction mixture was partitioned between ethyl acetate (100 mL) and anaqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 100 mL). Theorganic phase was separated and the aqueous phase was further extractedwith an additional 30-mL portion of ethyl acetate. The organic phaseswere combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, furnishing aclear oil. The product was purified by flash column chromatography(0.01:2:8 to 0.013:7 triethylamine-ethyl acetate-hexanes), affording theallylic sulfide MGC20 as a white foamy solid (728 mg, 97%).

R_(f) 0.65 (3:7 ethyl acetate-hexanes); ¹H NMR (400 MHz, C₆D₆) δ 7.35(d, 2H, J=7.2 Hz, o-ArH), 7.19 (m, 2H, o-ArH), 6.95 (m, 3H, p, m-ArH),6.89 (m, 2H, p, m-ArH), 6.83 (d, 1H, J=7.2 Hz, p-ArH), 5.51 (m, 1H,CH═CHCHSPh), 5.12 (m, 2H, CHOTBS, OCHH′Ph), 5.05 (d, 1H, J=12.4 Hz,OCHH′Ph), 4.73 (dt, 1H, J=10.0, 2.0 Hz, CH═CHCHSPh), 4.38 (m, 1H,CH═CHCHSPh), 3.47 (m, 1H, CHCHN(CH₃)₂), 2.92 (d, 1H, J=2.0 Hz,CHCHN(CH₃)₂), 1.75 (s, 6H, N(CH₃)₂), 1.14 (s, 9H, SiC(CH₃)₃), 0.35 (s,3H, SiCH₃), 0.31 (s, 3H, SiCH₃); ¹³C NMR (125 MHz, C₆D₆) δ 189.9, 177.0,168.9, 136.7, 135.2, 131.3, 130.3, 129.2, 128.5, 128.4, 128.3, 126.2,124.0, 106.2, 79.2, 72.4, 71.7, 63.2, 49.8, 43.4, 39.0, 26.6, 19.1,−2.9, −4.5; FTIR (neat), cm⁻¹ 3310 (m, OH), 2927 (m), 2854 (m), 2792(w), 1697 (s, C═O), 1621 (s), 1505 (s), 1470 (s), 1365 (s), 1254 (s),1145 (s), 1089 (s); HRMS (ES) m/z calcd for (C₃₂H₄₀N₂O₅SSi+H)⁺ 593.2505.found 593.2509.

Lower R_(f) Sulfoxide MGC21:

(−)-[(8,8)-(Dichlorocamphoryl)sunfonyl]oxaziridine (118 mg, 0.395 mmol,1.5 equiv) was added to a solution of the allylic sulfide MGC20 (156 mg,0.263 mmol, 1.0 equiv) in dichloromethane (2 mL) at 23° C. The mixturewas stirred at 23° C. for 20 h, then was concentrated, providing a lightbrown solid. The product was purified by flash column chromatography(0.001:2:8 to 0.001:3:7 triethylamine-ethyl acetate-hexanes), affordingthe lower R_(f) allylic sulfoxde MGC21 as a white solid (165 mg, 99%).

R_(f) 0.18 (3:7 ethyl acetate-hexanes); ¹H NMR (400 MHz, C₆D₆) δ 7.43(dd, 2H, J=8.0, 1.5 Hz, o-ArH), 7.16 (m, 2H, o-ArH), 6.92 (m, 6H, p,m-ArH), 5.43 (m, 1H, CH═CHCHS(O)Ph), 5.33 (d, 1H, J=5.0 Hz, CHOTBS),5.09 (d, 1H, J=11.5 Hz, OCHH′Ph), 5.02 (m, 2H, CH═CHCHS(O)Ph, OCHH′Ph),3.73 (m, 1H, CH═CHCHS(O)Ph), 3.41 (m, 1H, CHCHN(CH₃)₂), 2.85 (d, 1H,J=2.5 Hz, CHCHN(CH₃)₂), 1.70 (s, 6H, N(CH₃)₂), 1.12 (s, 9H, SiC(CH₃)₃),0.39 (s, 3H, SiCH₃), 0.36 (s, 3H, SiCH₃); ¹³C NMR (125 MHz, C₆D₆) δ189.5, 176.9, 168.8, 145.5, 135.2, 130.2, 129.9, 129.0, 128.5, 128.4,128.3, 127.8, 124.3, 122.9, 106.1, 79.3, 72.4, 70.6, 67.8, 63.1, 43.4,38.5, 26.6, 19.2, −2.6, −4.7; FTIR (neat), cm⁻¹ 3310 (m, OH), 2927 (m),2854 (m), 2792 (w), 1697 (s, C═O), 1621 (s), 1505 (s), 1470 (s), 1365(s), 1254 (s), 1145 (s), 1089 (s); HRMS (ES) m/z calcd for(C₃₂H₄₀N₂O₆SSi+H)⁺ 609.2455. found 609.2452.

Rearranged Allelic Alcohol MGC22:

Trimethylphosphite (0.620 mL, 5.26 mmol, 20.0 equiv) was added to asolution of the lower R_(f) allylic sulfoxide MGC21 (160 mg, 0.263 mmol,1.0 equiv) in methanol (5 mL) at 23° C. The solution was placed in anoil bath preheated to 65° C. and was stirred at that temperature for 36h. The solution was concentrated, providing a light yellow oil. Theproduct was purified by flash column chromatography (0.001:1:9 to0.001:2:8 triethylamine-ethyl acetate-hexanes), affording the allylicalcohol MGC22 as a white solid (100 mg, 76%). R_(f) 0.40 (3:7 ethylacetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.30 (d, 2H, J=7.0 Hz,o-ArH), 7.06 (dd, 2H, J=7.5, 7.0 Hz, m-ArH), 7.00 (d, 1H, J=7.5 Hz,p-ArH), 5.85 (m, 1H, ═CHCHOH), 5.42 (br d, 1H, J=10.5 Hz, =CHCHOTBS),5.16 (d, 1H, J=12.5 Hz, OCHH′Ph), 5.06 (d, 1H, J=12.5 Hz, OCHH′Ph), 4.44(m, 1H, ═CHCHOH), 4.31 (br s, 1H, OH), 4.07 (br s, 1H, =CHCHOTBS), 3.34(br s, 1H, OH), 3.33 (d, 1H, J=11.5 Hz, CHCHN(CH₃)₂), 2.75 (br d, 1H,J=11.5 Hz, CHCHN(CH₃)₂), 2.03 (s, 6H, N(CH₃)₂), 0.89 (s, 9H, SiC(CH₃)₃),−0.11 (s, 3H, SiCH₃), −0.13 (s, 3H, SiCH₃); ¹³C NMR (100 MHz, C₆D₆) δ189.7, 182.2, 167.7, 135.2, 129.2, 128.8, 128.3, 128.2, 106.6, 78.6,71.9, 68.1, 64.1, 59.6, 48.8, 41.2, 25.5, 17.8, −5.2, −5.6; FTIR (neat),cm⁻¹ 3515 (m, OH), 2917 (m), 2852 (m), 1708 (s, C═O), 1601 (s), 1511(s), 1471 (m), 1369 (m), 1254 (m), 1100 (m), 1022 (m); HRMS (ES) m/zcalcd for (C₂₆H₃₆N₂O₆Si+H)⁺501.2421. found 501.2424.

Benzyl Carbonate MGC23:

Benzyl chloroformate (120 μL, 0.841 mmol, 2.95 equiv) and4-(dimethylamino)pyridine (104 mg, 0.852 mmol, 3.0 equiv) were added insequence to a solution of the allylic alcohol MGC22 (142 mg, 0.284 mmol,1.0 equiv) in dichloromethane (3 mL) at 23° C. The reaction mixture wasstirred at 23° C. for 2 h, then was partitioned between ethyl acetate(50 mL) and saturated aqueous sodium bicarbonate solution (50 mL). Theorganic phase was separated and the aqueous phase was further extractedwith an additional 30-mL portion of ethyl acetate. The organic phaseswere combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing aclear oil (180 mg, 99%). The product was used in the next step withoutfurther purification. An analytical sample was prepared by purificationof the crude reaction mixture by flash column chromatography (0.001:2:8to 0.001:3:7 triethylamine-ethyl acetate-hexanes), affording the benzylcarbonate MGC23 as a white solid.

R_(f) 0.60 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.26(d, 2H, J=7.0 Hz, o-ArH), 7.02 (m, 8H, ArH), 5.75 (br dd, 1H, J=10.5,3.0 Hz, =CHCHOCO₂Bn), 5.70 (br dd, 1H, J=10.5, 2.5 Hz, =CHCHOTBS), 5.37(m, 1H, =CHCHOCO₂Bn), 5.10 (d, 1H, J=12.5 Hz, OCHH′Ph), 5.06 (d, 1H,J=12.5 Hz, OCHH′Ph), 4.91 (d, 1H, J=12.0 Hz, OCHH′Ph′), 4.88 (d, 1H,J=12.0 Hz, OCHH′Ph′), 4.41 (m, 1H, =CHCHOTBS), 3.38 (d, 1H, J=7.5 Hz,CHCHN(CH₃)₂), 3.11 (m, 1H, CHCHN(CH₃)₂), 1.92 (s, 6H, N(CH₃)₂), 0.92 (s,9H, SiC(CH₃)₃), 0.02 (s, 3H, SiCH₃), −0.02 (s, 3H, SiCH₃); ¹³C NMR (100MHz, C₆D₆) δ 188.9, 179.9, 168.3, 155.2, 135.6, 135.4, 133.2, 128.6,128.5, 128.4, 128.3, 127.7, 124.9, 107.0, 77.3, 72.2, 71.6, 69.6, 66.6,60.3, 44.4, 42.2, 25.9, 18.2, −4.8, −4.8; FTIR (neat), cm⁻¹ 3532 (w,OH), 2948 (m), 2842 (m), 1738 (s, C═O), 1708 (s, C═O), 1608 (s), 1512(s), 1471 (m), 1383 (m), 1258 (s), 1101 (m); HRMS (ES) m/z calcd for(C₃₄H₄₂N₂O₈Si+H)⁺ 635.2789. found 635.2786.

Diol MGC24:

Acetic acid (40.0 μL, 0.709 mmol, 2.5 equiv) and a solution oftetrabutylammonium fluoride in tetrahydrofuran (1.0 M, 0.709 mL, 0.709mmol, 2.5 equiv) were added in sequence to a solution of the benzylcarbonate MGC23 (180 mg, 0.284 mmol, 1.0 equiv) in tetrahydrofuran (3mL) at 23° C. The resulting yellow solution was stirred at 23° C. for 4h, then was partitioned between ethyl acetate (50 mL) and an aqueouspotassium phosphate buffer solution (pH 7.0, 0.2 M, 50 mL). The organicphase was separated and the aqueous phase was further extracted with two20-mL portions of ethyl acetate. The organic phases were combined anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a brown oil. The product waspurified by flash column chromatography (2:8 to 1:1 ethylacetate-hexanes), affording the diol MGC24 as a white solid (135 mg, 92%over 2 steps).

R_(f) 0.15 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 7.24(d, 2H, J=7.0 Hz, o-ArH), 7.02 (m, 8H, ArH), 5.68 (br dd, 1H, J=10.5,2.5 Hz, =CHCHOCO₂Bn), 5.63 (br dd, 1H, J=10.5, 3.0 Hz, ═CHCHOH), 5.26(m, 1H, =CHCHOCO₂Bn), 5.09 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.05 (d, 1H,J=12.0 Hz, OCHH′Ph), 4.89 (d, 1H, J=12.0 Hz, OCHH′Ph′), 4.86 (d, 1H,J=12.0 Hz, OCHH′Ph′), 4.16 (m, 1H, ═CHCHOH), 3.24 (d, 1H, J=6.5 Hz,CHCHN(CH₃)₂), 2.94 (m, 1H, CHCHN(CH₃)₂), 2.25 (br s, 1H, OH), 1.82 (s,6H, N(CH₃)₂)_(;) ¹³C NMR (100 MHz, CDCl₃) δ 168.1, 154.8, 135.1, 134.9,132.2, 128.9, 128.9, 128.8, 128.7, 128.6, 126.4, 106.7, 76.6, 72.9,71.3, 70.3, 64.9, 60.3, 44.4, 43.3; FTIR (neat), cm⁻¹ 3468 (m, OH), 3034(w), 2949 (m), 2798 (m), 1738 (s, C═O), 1705 (s, C═O), 1606 (s), 1513(s), 1475 (m), 1379 (m), 1261 (s), 1022 (m); HRMS (ES) m/z calcd for(C₂₈H₂₈N₂O₈+H)⁺ 521.1929. found 521.1926.

Cyclohexenone MGC25:

Solid o-iodoxybenzoic acid (79.0 mg, 0.281 mmol, 6.5 equiv) was added inone portion to a solution of the diol MGC24 (22.5 mg, 0.0433 mmol, 1.0equiv) in dimethylsulfoxide (0.7 mL) at 23° C. The reaction mixture wasinitially heterogeneous, but became homogeneous within 5 min. The brownreaction mixture was protected from light and was stirred vigorously at23° C. for 12 h. The resulting orange reaction mixture was partitionedbetween ether (20 mL) and water (20 mL). The organic phase was separatedand the aqueous phase was further extracted with two 10 mL-portionsether. The organic phases were combined and washed with saturatedaqueous sodium bicarbonate solution (8 mL, containing 30 mg of sodiumbisulfite) and brine (10 mL). The washed solution was dried overanhydrous sodium sulfate and filtered. The filtrate was concentrated,yielding the cyclohexenone MGC25 as a white oily solid (22.2 mg, 99%).

R_(f) 0.33 (2:3 ethyl acetate-hexanes); ¹H NMR (400 MHz, C₆D₆) δ 7.22(d, 2H, J=6.8 Hz, o-ArH), 6.99 (m, 8H, ArH), 6.12 (ddd, 1H, J=10.4, 4.0,1.2 Hz, CH═CHCHOCO₂Bn), 5.74 (dd, 1H, J=10.4, 1.2 Hz, CH═CHCHOCO₂Bn),5.41 (ddd, 1H, J=4.0, 1.2, 1.2 Hz, CH═CHCHOCO₂Bn), 5.18 (br s, 1H, OH),5.08 (d, 1H, J=12.0 Hz, OCHH′Ph), 5.01 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.89(d, 1H, J=12.4 Hz, OCHH′Ph′), 4.83 (d, 1H, J=12.4 Hz, OCHH′Ph′), 3.28(d, 1H, J=8.4 Hz, CHCHN(CH₃)₂), 2.85 (ddd, 1H, J=8.4, 4.0, 1.2 Hz,CHCHN(CH₃)₂), 1.92 (s, 6H, N(CH₃)₂); ¹³C NMR (100 MHz, C₆D₆) δ 192.3,186.2, 180.5, 167.8, 154.8, 141.8, 135.3, 135.2, 129.9, 128.6, 128.6,128.5, 128.4, 127.8, 107.7, 78.9, 72.5, 69.9, 59.9, 48.4, 41.9; FTIR(neat), cm⁻¹ 3442 (m, OH), 3030 (w), 2948 (m), 2793 (m), 1742 (s, C═O),1711 (s, C═O), 1608 (s), 1510 (s), 1448 (m), 1376 (m), 1258 (s), 1056(m); HRMS (ES) m/z calcd for (C₂₈H₂₆N₂O₈+H)⁺ 519.1767. found 519.1773.

Silyl-Cyclohexenone MGC26:

Triethylamine (172 μL, 1.24 mmol, 3.5 equiv) and tert-butyldimethylsilyltrifluoromethanesulfonate (243 μL, 1.06 mmol, 3.0 equiv) were added insequence to a solution of the cyclohexenone MGC25 (183 mg, 0.353 mmol,1.0 equiv) in tetrahydrofuran (8 mL) at 0° C. The reaction mixture wasstirred at 0° C. for 40 min, then was partitioned between ethyl acetate(50 mL) and an aqueous potassium phosphate buffer solution (pH 7.0, 0.2M, 50 mL). The organic phase was separated and the aqueous phase wasfurther extracted with a 25-mL portion of ethyl acetate. The organicphases were combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing ayellow oily solid. The product was purified by flash columnchromatography (1:9 to 2:8 ethyl acetate-hexanes), affording thesilyl-cyclohexenone MGC26 as a clear oil (207 mg, 93%).

R_(f) 0.50 (3:7 ethyl acetate-hexanes); ¹H NMR (400 MHz, C₆D₆) δ 7.21(dd, 2H, J=7.5, 1.0 Hz, o-ArH), 7.15 (d, 2H, J=8.0 Hz, o-ArH), 7.05 (t,2H, J=8.0 Hz, m-ArH), 6.98 (m, 4H, m, p-ArH), 6.30 (ddd, 1H, J=10.5,5.0, 2.0 Hz, CH═CHCHOCO₂Bn), 5.68 (dd, 1H, J=10.5, 1.0 Hz,CH═CHCHOCO₂Bn), 5.65 (br d, 1H, J=5.0 Hz, CH═CHCHOCO₂Bn), 5.10 (d, 1H,J=12.5 Hz, OCHH′Ph), 5.01 (d, 1H, J=12.5 Hz, OCHH′Ph), 4.95 (d, 1H,J=12.5 Hz, OCHH′Ph′), 4.82 (d, 1H, J=12.5 Hz, OCHH′Ph′), 3.11 (d, 1H,J=11.0 Hz, CHCHN(CH₃)₂), 2.94 (br d, 1H, J=11.0 Hz, CHCHN(CH₃)₂), 1.96(s, 6H, N(CH₃)₂), 1.08 (s, 9H, SiC(CH₃)₃), 0.59 (s, 3H, SiCH₃), 0.29 (s,3H, SiCH₃); ¹³C NMR (100 MHz, C₆D₆) δ 193.3, 186.7, 180.3, 167.8, 154.9,140.9, 135.6, 135.3, 129.9, 128.6, 128.5, 128.5, 128.4, 128.0, 127.8,108.6, 82.4, 72.4, 69.6, 69.3, 59.7, 50.2, 41.4, 26.5, 19.6, −1.9, −3.4;FTIR (neat), cm⁻¹ 2930 (m), 2855 (m), 1745 (s, C═O), 1722 (s, C═O), 1691(m), 1613 (m), 1513 (s), 1473 (m), 1455 (m), 1378 (m), 1264 (s), 1231(s), 1046 (m); HRMS (ES) m/z calcd for (C₃₄H₄₀N₂O₈+H)⁺ 633.2632. found633.2620.

Michael-Dieckmann Addition Product MGC27:

A solution of n-butyllithium in hexanes (1.55 M, 155 μL, 0.241 mmol, 5.1equiv) was added to a solution of N,N,N′,N′-tetramethylethylenediamine(39.0 μL, 0.261 mmol, 5.5 equiv) and diisopropyl amine (34.0 μL, 0.249mmol, 5.25 equiv) in tetrahydrofuran (1 mL) at −78° C. The resultingmixture was stirred vigorously at −78° C. for 30 min whereupon asolution of the ester CDL-I-280 (73.0 mg, 0.213 mmol, 4.5 equiv) intetrahydrofuran (1 mL) was added dropwise via cannula. The resultingdeep red mixture was stirred vigorously at −78° C. for 75 min, then asolution of the silyl-cyclohexenone MGC26 (30.0 mg, 0.0474 mmol, 1.0equiv) in tetrahydrofuran (1 mL) was added dropwise via cannula. Theresulting light red mixture was allowed to warm slowly to 0° C. over 2h, then was partitioned between an aqueous potassium phosphate buffersolution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The organicphase was separated and the aqueous phase was further extracted with two10-mL portions of dichloromethane. The organic phases were combined anddried over anhydrous sodium sulfate. The dried solution was filtered andthe filtrate was concentrated, providing a yellow oil. The product waspurified by preparatory HPLC on a Coulter Ultrasphere ODS column (10 μM,250×10 mm, flow rate 3.5 mL/min, Solvent A: methanol, Solvent B: water)using an injection volume of 400 μL (methanol) and an isochratic elutionof 10% B for 75 min. The peak eluting at 36-42 min was collected andconcentrated, affording the Michael-Dieckmann addition product MGC27(33.0 mg, 80%) as a light yellow solid.

R_(f) 0.35 (1:4 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 16.55(br s, 1H, enol), 7.26 (d, 2H, J=7.0 Hz, o-ArH), 7.14 (d, 2H, J=7.5 Hz,ArH), 6.85-7.05 (m, 6H, ArH), 6.66-6.74 (m, 2H, ArH), 6.51 (dd, 1H,J=9.0, 1.5 Hz, ArH), 5.73 (br d, 1H, J=4.0 Hz, BnOCO₂CH), 5.17 (d, 1H,J=12.5 Hz, OCHH′Ph), 5.03 (d, 1H, J=12.5 Hz, OCHH′Ph), 4.99 (d, 1H,J=12.5 Hz, OCHH′Ph′), 4.93 (d, 1H, J=12.5 Hz, OCHH′Ph′), 3.58 (d, 1H,J=11.5 Hz, CHCHN(CH₃)₂), 3.35 (dd, 1H, J=12.5, 4.0 Hz, CH₃CHCH), 2.99(d, 1H, J=11.5 Hz, CHCHN(CH₃)₂), 2.56 (dq, 1H, J=12.5, 7.0 Hz, CH₃CH),2.18 (s, 6H, N(CH₃)₂), 1.33 (s, 9H, C(CH₃)₃), 1.16 (d, 3H, J=7.0 Hz,CH₃CH), 1.11 (s, 9H, C(CH₃)₃), 0.61 (s, 3H, CH₃), 0.36 (s, 3H, CH₃); ¹³CNMR (100 MHz, CDCl₃) δ 189.7, 186.3, 180.9, 178.4, 167.9, 154.7, 152.1,150.8, 145.9, 136.1, 135.5, 133.9, 128.7, 128.6, 128.5, 127.3, 123.8,122.7, 122.6, 108.9, 105.5, 83.0, 82.9, 74.8, 72.4, 69.2, 60.8, 52.7,43.2, 38.4, 27.5, 26.6, 19.5, 16.3, −1.8, −2.7; FTIR (neat film), cm⁻¹2974 (w), 2933 (w), 2851 (w), 1760 (s, C═O), 1748 (s, C═O), 1723 (s,C═O), 1606 (m), 1513 (m), 1471 (m), 1370 (m). 1260 (s), 1232 (s), 1148(s); HRMS (ES) m/z calcd for (C₄₈H₅₆O₁₂N₂Si)⁺ 881.3681. found 881.3684.

Initial Deprotection of Michael-Dieckmann Addition Product MGC28:

Hydrofluoric acid (1.2 mL, 48% aqueous) was added to a polypropylenereaction vessel containing a solution of the Michael-Dieckmann additionproduct MGC27 (33.0 mg, 0.0375 mmol, 1.0 equiv) in acetonitrile (7.0 mL)at 23° C. The resulting mixture was stirred vigorously at 23° C. for 60h, then was poured into water (50 mL) containing K₂HPO₄ (7.0 g). Theresulting mixture was extracted with ethyl acetate (3×20 mL). Theorganic phases were combined and dried over anhydrous sodium sulfate.The dried solution was filtered and the filtrate was concentrated,furnishing the pentacyclic phenol MGC28 as a yellow oil (25.0 mg, 99%).The product was used in the next step without further purification.

R_(f) 0.05 (1:4 ethyl acetate-hexanes); ¹H NMR (600 MHz, C₆D₆, crude) δ14.86 (br s, 1H, enol), 11.95 (s, 1H, phenol), 7.23 (d, 2H, J=7.8 Hz,o-ArH), 7.14 (d, 2H, J=7.2 Hz, o-ArH), 6.94-7.02 (m, 6H, ArH), 6.86 (t,1H, J=8.4 Hz, ArH), 6.76 (d, 1H, J=8.4 Hz, ArH), 6.28 (d, 1H, J=7.8 Hz,ArH), 5.46 (dd, 1H, J=3.6, 3.0 Hz, BnOCO₂CH), 5.12 (d, 1H, J=12.0 Hz,OCHH′Ph), 5.04 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.92 (s, 2H, OCH₂Ph), 3.41(d, 1H, J=9.6 Hz, CHCHN(CH₃)₂), 2.82 (dd, 1H, J=9.6, 3.0 Hz,CHCHN(CH₃)₂), 2.65 (dd, 1H, J=13.2, 3.6 Hz, CH₃CHCH), 2.78 (dq, 1H,J=13.2, 7.2 Hz, CH₃CH), 2.05 (s, 6H, N(CH₃)₂), 1.04 (d, 3H, J=7.2 Hz,CH₃CH); ¹³C NMR (100 MHz, C₆D₆, crude) δ 193.4, 186.2, 181.3, 172.3,167.9, 163.3, 154.6, 145.8, 136.6, 135.8, 128.6, 128.4, 127.2, 116.8,116.0, 115.6, 107.6, 104.7, 76.8, 73.9, 72.5, 69.5, 60.3, 48.7, 43.0,41.8, 37.5, 15.3; FTIR (neat film), cm⁻¹ 3424 (m, OH), 3059, 3030, 2925,2857, 1744 (s, C═O), 1713 (s, C═O), 1614 (s), 1582 (s), 1455 (s), 1252(s); HRMS (ES) m/z calcd for (C₃₇H₃₄O₁₀N₂+H)⁺ 667.2292. found 667.2300.

(−)-Doxycycline (MGC30):

Pd black (7.00 mg, 0.0657 mmol, 1.75 equiv) was added in one portion toa solution of the pentacyclic phenol MGC28 (25.0 mg, 0.0375 mmol, 1.0equiv) in tetrahydrofuran-methanol (1:1, 2.0 mL) at 23° C. An atmosphereof hydrogen was introduced by briefly evacuating the flask, thenflushing with pure hydrogen (1 atm). The Pd catalyst was initiallypresent as a fine dispersion, but aggregated into clumps within 5 min.The yellow heterogeneous mixture was stirred at 23° C. for 2 h, then wasfiltered through a plug of cotton. The filtrate was concentrated,affording a yellow oil (>95% doxycycline based on ¹H NMR analysis). Theproduct was purified by preparatory HPLC on a Phenomenex Polymerx DVBcolumn (10 μM, 250×10 mm, flow rate 4.0 mL/min, Solvent A:methanol-0.005 N aq. HCl (1:4), Solvent B: acetonitrile) using aninjection volume of solvent A (400 μL) containing oxalic acid (10 mg)and an isochratic elution of 5% B for 2 min, then a gradient elution of5-50% B for 20 min. The peak eluting at 12-17 min was collected andconcentrated, affording (−)-doxycycline hydrochloride as a yellow powder(16.2 mg, 90%), which was identical with natural (−)-doxycyclinehydrochloride in all respects.

¹H NMR (600 MHz, CD₃OD, hydrochloride) δ 7.47 (t, 1H, J=8.4 Hz, ArH),6.93 (d, 1H, J=8.4 Hz, ArH), 6.83 (d, 1H, J=8.4 Hz, ArH), 4.40 (s, 1H,(CH₃)₂NCH), 3.53 (dd, 1H, J=12.0, 8.4 Hz, CHOH), 2.95 (s, 3H,N(CH₃)CH₃′), 2.88 (s, 3H, N(CH₃)CH₃′), 2.80 (d, 1H, J=12.0 Hz,CHCHN(CH₃)₂), 2.74 (dq, 1H, J=12.6, 6.6 Hz, CH₃CH), 2.58 (dd, 1H,J=12.6, 8.4 Hz, CH₃CHCH), 1.55 (d, 3H, J=6.6 Hz, CH₃CHCH); ¹³C NMR (100MHz, CD₃OD) δ 195.3, 188.2, 173.8, 172.1, 163.2, 149.0, 137.7, 117.1,116.9, 116.6, 108.4, 96.0, 74.5, 69.8, 66.9, 47.5, 43.4, 43.0, 41.9,40.0, 16.3; UV max (0.01 N methanolic HCl), nm 218, 267, 350;[α]_(D)=−109° (c=0.16 in 0.01 M methanolic HCl); lit. (The Merck Index:An Encyclopedia of Chemicals, Drugs, and Biologicals, 12^(th) ed.Budavari, S.; O'Neal, M. J.; Smith, A.; Heckelman, P. E.; Kinneary, J.F., Eds.; Merck & Co.: Whitehouse Station, N J, 1996; entry 3496.) UVmax (0.01 N methanolic HCl), nm 267, 351; [α]_(D)=−110° (c=1 in 0.01 Mmethanolic HCl); HRMS (ES) m/z calcd for (C₂₂H₂₄O₈N₂+H)⁺ 445.1611. found445.1603.

Example 3—Synthesis of 6-Deoxytetracycline

Ester CDL-I-280:

A solution of sec-butyllithium in cyclohexane (1.40 M, 24.0 mL, 33.6mmol, 2.6 equiv) was added to a solution ofN,N,N′,N′-tetramethylethylenediamine (4.9 mL, 33 mmol, 2.5 equiv) intetrahydrofuran (25 mL) at −78° C. The resulting yellow solution wascooled to −90° C. (internal temperature) in a liquid nitrogen-ethanolbath. A solution of o-anisic acid (2.00 g, 13.1 mmol, 1.0 equiv) intetrahydrofuran (10 mL) was added dropwise via cannula over a period of30 min to the yellow solution. The resulting orange suspension wasstirred for an additional 30 min at −90° C., then was allowed to warm to−78° C. over 15 min, whereupon iodoethane (4.2 mL, 52 mmol, 4.0 equiv)was added. The mixture was allowed to warm to 23° C. over 15 min, thenwas partitioned between water (50 mL) and ether (50 mL). The aqueouslayer was separated and diluted with aqueous hydrochloric acid (1.0 M,100 mL). The resulting mixture was extracted with ethyl acetate (4×80mL). The organic layers were combined and then dried over anhydroussodium sulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a brown oil (1.8 g). ¹H NMR (500 MHz, CDCl₃)analysis of the crude product showed an 8:2 ratio of the carboxylic acidCDL-I-279 (δ 3.89, OCH₃) and unreacted anisic acid (δ 4.07, OCH₃).Oxalyl chloride (1.0 mL, 11 mmol, 0.8 equiv) and N,N-dimethylformamide(100 μL) were added in sequence to a solution of the residue indichloromethane (20 mL) at 23° C. Vigorous gas evolution was observedupon addition of N,N-dimethylformamide. The reaction mixture was stirredfor 2 h at 23° C., whereupon phenol (1.4 g, 15 mmol, 1.1 equiv),pyridine (2.4 mL, 30 mmol, 2.3 equiv), and 4-(dimethylamino)pyridine (10mg, 0.081 mmol, 0.006 equiv) were added in sequence at 23° C. Theresulting brown reaction mixture was then stirred for 2 h at 23° C.Aqueous hydrochloric acid (1 M, 50 mL) was added and the resultingmixture was extracted with ethyl acetate (2×50 mL). The organic layerswere combined, then washed with an aqueous sodium hydroxide solution(0.1 M, 50 mL), followed by brine (50 mL), and were then dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a clear oil. The product waspurified by flash column chromatography (5:95 ethyl acetate-hexanes),affording the ester CDL-I-280 as a colorless oil (1.7 g, 50%).

R_(f) 0.28 (5:95 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.56(t, 2H, J=7.8 Hz, ArH), 7.37 (t, 1H, J=7.8 Hz, ArH), 7.31-7.26 (m, 3H,ArH), 6.93 (d, 1H, J=7.8 Hz, ArH), 6.85 (d, 1H, J=8.3 Hz, ArH), 3.91 (s,3H, OCH₃), 2.79 (q, 2H, J=7.8 Hz, CH₂CH₃), 1.33 (t, 3H, J=7.8 Hz,CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 166.9, 156.5, 150.8, 142.8, 130.9,129.5, 125.9, 122.5, 121.6, 120.9, 108.5, 55.9, 26.6, 15.6; FTIR (neatfilm), cm⁻¹ 2970 (m), 1740 (s, C═O), 1583 (s), 1488 (s), 1471 (s), 1438(m), 1298 (w), 1270 (s), 1236 (s), 1186 (s), 1158 (m), 1091 (m), 1046(s), 1001 (w); HRMS (ES) m/z calcd for (C₁₆H₁₆O₃+H)⁺ 257.1178. found257.1183.

Phenol CDL-I-298:

A solution of boron tribromide in dichloromethane (1.0 M, 5.2 mL, 5.2mmol, 2.0 equiv) was added to a solution of the ester CDL-I-280 (662 mg,2.58 mmol, 1.0 equiv) in dichloromethane (10 mL) at 0° C. The resultingyellow solution was stirred for 70 min at 0° C., whereupon saturatedaqueous sodium bicarbonate solution (50 mL) was added. The resultingbiphasic mixture was stirred for 20 min at 0° C., dichloromethane (50mL) was added, the layers were separated, and the aqueous phase wasfurther extracted with dichloromethane (50 mL). The organic layers werecombined and then dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing thephenol CDL-I-298 as a colorless oil (605 mg, 97%).

R_(f) 0.47 (5:95 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 10.94(s, 1H, OH), 7.49 (t, 2H, J=7.8 Hz, ArH), 7.41 (t, 1H, J=7.8 Hz, ArH),7.35 (t, 1H, J=7.3 Hz, ArH), 7.24 (d, 2H, J=7.8 Hz, ArH), 6.93 (d, 1H,J=8.3 Hz, ArH), 6.85 (d, 1H, J=8.3 Hz, ArH), 3.13 (q, 2H, J=7.8 Hz,CH₂CH₃), 1.34 (t, 3H, J=7.8 Hz, CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ170.3, 163.2, 149.8, 147.8, 135.1, 129.7, 126.4, 122.0, 121.6, 115.9,111.1, 29.8, 16.4; FTIR (neat film), cm⁻¹ 2973 (w), 1670 (s, C═O), 1609(m), 1588 (m), 1490 (w), 1444 (m), 1311 (m), 1295 (m), 1234 (m), 1187(s), 1162 (s), 1105 (m); HRMS (ES) m/z calcd for (C₁₅H₁₄O₃+H)⁺ 243.1021.found 243.1014.

Ester CDL-I-299:

N,N-diisopropylethylamine (520 μL, 2.99 mmol, 1.2 equiv), di-t-butyldicarbonate (645 mg, 2.96 mmol, 1.2 equiv), and4-(dimethylamino)pyridine (31 mg, 0.25 mmol, 1.5 equiv) were added insequence to a solution of the phenol CDL-I-298 (605 mg, 2.50 mmol, 0.1equiv) in dichloromethane (10 mL) at 23° C. The reaction mixture wasstirred for 1 h at 23° C., whereupon saturated aqueous ammonium chloridesolution (50 mL) was added. Dichloromethane (50 mL) was added, thelayers were separated, and the aqueous phase was extracted withdichloromethane (50 mL). The organic layers were combined and then driedover sodium sulfate. The dried solution was filtered and the filtratewas concentrated, providing a brown oil. The product was purified byflash column chromatography (1:9 ether-hexanes), affording the esterCDL-I-299 as a colorless oil, which crystallized upon standing overnightat −14° C. (733 mg, 86%), mp 58° C.

R_(f) 0.23 (1:9 ether-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.46-7.42 (m,3H, ArH), 7.31-7.26 (m, 3H, ArH), 7.22 (d, 1H, J=7.3 Hz, ArH), 7.15 (d,1H, J=7.3 Hz, ArH), 2.86 (q, 2H, J=7.3 Hz, CH₂CH₃), 1.46 (s, 9H, Boc),1.31 (t, 3H, J=7.3 Hz, CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 165.1, 151.6,150.6, 148.7, 144.5, 131.3, 129.4, 126.8, 126.1, 125.4, 121.7, 120.5,83.8, 27.5, 26.8, 15.6; FTIR (neat film), cm⁻¹ 2964 (w), 1754 (s, C═O),1586 (w), 1491 (w), 1467 (w), 1457 (w), 1368 (w), 1278 (s), 1234 (s),1190 (s), 1145 (s), 1051 (m); HRMS (ES) m/z calcd for (C₂₀H₂₂O₅+NH₄)⁺360.1811. found 360.1808.

Michael-Dieckmann Addition Product CDL-I-287:

A solution of n-butyllithium in hexanes (1.45 M, 47 μL, 0.068 mmol, 6.8equiv) was added to a solution of diisopropylamine (10 μL, 0.071 mmol,7.1 equiv) and N,N,N′,N′-tetramethylethylenediamine (10 μL, 0.066 mmol,6.6 equiv) in tetrahydrofuran (300 μL) at −78° C. The resulting solutionwas stirred at −78° C. for 30 min whereupon a solution of the esterCDL-I-299 (17 mg, 0.050 mmol, 5.0 equiv) in tetrahydrofuran (200 μL) wasadded, forming a deep red solution. The solution was stirred at −78° C.for 75 min, then a solution of the enone DRS6 (5.0 mg, 0.010 mmol, 1.0equiv) in tetrahydrofuran (100 μL) was added at −78° C. The color of thereaction mixture remained deep red following the addition. The mixturewas allowed to warm to 0° C. over 150 min. Upon reaching 0° C., anaqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) wasadded. The resulting yellow mixture was extracted with dichloromethane(3×15 mL). The organic layers were combined and then dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, providing a yellow oil. The product waspurified by preparatory HPLC on a Coulter Ultrasphere ODS column (5 μm,250×10 mm, flow rate 3.5 mL/min, Solvent A: water, Solvent B: methanol,UV detection at 350 nm) using an injection volume of 500 μL methanolwith an isochratic elution of 89.5% B. The peak eluting at 31-40 min wascollected and concentrated affording the Michael-Dieckmann productCDL-I-287 as a light yellow solid (6.1 mg, 83%), mp 114° C.

R_(f) 0.37 (2:8 tetrahydrofuran-hexanes); ¹H NMR (500 MHz, CDCl₃) δ (s,1H, 16.24, enol-OH), 7.55-7.50 (m, 3H, ArH), 7.40-7.35 (m, 4H, ArH),7.10 (d, 1H, J=7.8 Hz, ArH), 5.39-5.34 (m, 2H, OCH₂Ph), 3.92 (d, 1H,J=10.7 Hz, CHN(CH₃)₂), 2.81-2.71 (m, 2H, CH₃CH, CH₃CHCH), 2.55 (dd, 1H,J=10.7, 5.7 Hz, CHCHN(CH₃)₂), 2.48 (s, 6H, N(CH₃)₂), 2.40 (d, 1H, J=14.7Hz, CHH′CHCHN(CH₃)₂), 2.31 (ddd, 1H, J=14.7, 9.3, 5.7, CHH′CHCHN(CH₃)₂),1.56 (s, 3H, CH₃), 1.55 (s, 9H, Boc), 0.84 (s, 9H, TBS), 0.27 (s, 3H,TBS), 0.13 (s, 3H, TBS); ¹³C NMR (125 MHz, CDCl₃) δ 187.4, 183.1, 182.8,181.6, 167.6, 151.7, 150.2, 147.4, 135.0, 134.0, 128.5, 128.5, 123.4,123.0, 122.4, 108.3, 107.4, 94.8, 83.9, 81.5, 72.5, 61.5, 46.4, 41.9,39.5, 34.9, 27.7, 26.0, 20.7, 19.0, 16.0, −2.6, −3.7; FTIR (neat film),cm⁻¹ 2923 (m), 2841 (m), 1759 (s, C═O), 1718 (s, C═O), 1605 (s), 1508(s), 1467 (m), 1456 (m), 1369 (m), 1277 (s), 1262 (m), 1231 (s), 1144(s), 1005 (w); HRMS (ES) m/z calcd for (C₄₀H₅₀N₂O₉Si+H)⁺ 731.3364. found731.3370.

6-Deoxytetracycline CDL-I-322

Hydrofluoric acid (0.6 mL, 48% aqueous) was added to a polypropylenereaction vessel containing a solution of the Michael-Dieckmann additionproduct CDL-I-287 (15 mg, 0.021 mmol, 1.0 equiv) in acetonitrile (3.5mL) at 23° C. The reaction mixture was stirred at 23° C. for 55 h, thenwas poured into water (20 mL) containing K₂HPO₄ (4.0 g). The resultingmixture was extracted with ethyl acetate (4×20 mL). The organic phaseswere combined and then dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing alight yellow oil. Pd black (7.6 mg, 0.071 mmol, 3.4 equiv) was added inone portion to a solution of the residue in methanol-tetrahydrofuran(1:1, 2 mL). An atmosphere of hydrogen gas was introduced by brieflyevacuating the flask, then flushing with pure hydrogen (1 atm). Themixture was stirred at 23° C. for 2 h. Within 5 min, the color changedfrom light yellow to dark yellow. The reaction mixture was filteredthrough a plug of cotton. The filtrate was concentrated, affording ayellow oil (10 mg). The product was purified by preparatory HPLC on aPhenomenex Polymerx DVB column (10 μm, 250×10 mm, flow rate 5 mL/min,Solvent A: methanol-0.02 N HCl (1:4), Solvent B: acetonitrile, UVdetection at 365 nm) using an injection volume of 400 μL methanolcontaining oxalic acid monohydrate (10 mg) and an isochratic elution of18% B for 15 min, then a linear gradient elution of 18-60% B in 15 min.The peak eluting at 17.5-22.5 min was collected and concentrated to give6-deoxytetracycline hydrochloride (CDL-I-322.HCl) as a yellow powder(8.1 mg, 81%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 7.49 (t, 1H, J=7.8 Hz, ArH),6.95 (d, 1H, J=7.8 Hz, ArH), 6.84 (d, 1H, J=7.8 Hz, ArH), 4.09 (s, 1H,CHN(CH₃)₂), 3.03 (br s, 3H, N(CH₃)), 2.97 (br s, 3H, N(CH₃)), 2.90 (brd, 1H, J=12.7 Hz, CHCHN(CH₃)₂), 2.67 (ddd, 1H, J=12.7, 12.7, 5.2 Hz,CH₃CHCH), 2.61-2.56 (m, 1H, CH₃CH), 2.30 (ddd, J=13.7, 5.2, 2.9 Hz,CHH′CHCHN(CH₃)₂), 1.54 (ddd, J=13.7, 12.7, 12.7 Hz, CHH′CHCHN(CH₃)₂),1.38 (d, 3H, J=6.8 Hz, CH₃CH). HRMS (ES) m/z calcd for (C₂₂H₂₄N₂O₇+H)⁺429.1662. found 429.1660.

Example 4—Synthesis of a Pyridone Sancycline Analog

Phenyl Ester CDL-II-464:

2,4,6-Trichlorobenzoyl chloride (356 μL, 2.28 mmol, 1.1 equiv) was addedto a solution of the carboxylic acid CDL-II-417 (reported by A. N.Osman, M. M Ismail, M. A. Barakat, Revue Roumaine de Chime 1986, 31,615-624) (534 mg, 2.08 mmol. 1.0 equiv) and triethylamine (320 μL, 2.28mmol, 1.1 equiv) in tetrahydrofuran (25 mL) at 23° C. A whiteprecipitate was formed upon addition. The reaction mixture was stirredfor 30 min at 23° C. A solution of phenol (489 mg, 5.20 mmol, 2.5 equiv)and 4-(dimethylamino)pyridine (583 mg, 5.20 mmol, 2.5 equiv) intetrahydrofuran (10 mL) was added via cannula to the reaction mixtureprepared above at 0° C. The resulting mixture was allowed to warm to 23°C. over 10 min, and was stirred for 90 min at that temperature. Anaqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) wasthen added and the resulting mixture was extracted with dichloromethane(3×30 mL). The organic extracts were combined and then dried overanhydrous sodium sulfate. The dried solution was filtered and thefiltrate concentrated, providing a colorless oil. The product waspurified by flash column chromatography (6:94 ethyl acetate-hexanes),affording the phenyl ester CDL-II-464 as a white solid (590 mg, 85%), mp65° C.

R_(f) 0.33 (1:9 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 7.49(d, 2H, J=7.3 Hz, ArH), 7.40-7.24 (m, 6H, ArH), 7.14 (d, 2H, J=7.3 Hz,ArH), 6.69 (s, 1H, pyr-H), 5.49 (s, 2H, CH₂Ph), 2.47 (s, 3H, CH₃), 2.43(s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 165.9, 160.1, 157.8, 150.7,148.5, 137.3, 129.4, 128.3, 127.7, 127.6, 125.9, 121.7, 118.1, 113.4,67.8, 24.1, 19.2; FTIR (neat film), cm⁻¹ 1738 (s, C═O), 1600 (s), 1569(s), 1492 (m), 1441 (m), 1400 (m), 1333 (s), 1272 (s), 1185 (s), 1159(m), 1097 (m), 1051 (s); HRMS (ES) m/z calcd for (C₂₁H₁₉NO₃+H)⁺334.1443. found 334.1442.

Michael-Dieckmann Addition Product CDL-II-466:

A solution of n-butyllithium in hexanes (1.67 M, 80 μL, 0.13 mmol, 4.2equiv) was added to a solution of diisopropylamine (20 μL, 0.14 mmol,4.5 equiv) in tetrahydrofuran (2.5 mL) at −78° C. The resulting solutionwas allowed to warm to 0° C. over 15 min. N,N′-dimethylpropyleneurea (17μL, 0.14 mmol, 4.5 equiv) was added to the mixture prepared above at 0°C., whereupon the mixture was cooled to −78° C. A solution of the esterCDL-II-464 (31 mg, 0.093 mmol, 3.0 equiv) in tetrahydrofuran (250 μL)was then added at −78° C. The resulting yellow solution was stirred for5 min at −78° C., then a solution of the enone DRS6 (15 mg, 0.031 mmol,1.0 equiv) in tetrahydrofuran (250 μL) was added at −78° C. Theresulting deep red mixture was allowed to warm to 0° C. over 4 h. Aceticacid (40 μL) was added at to the deep red mixture at 0° C., followed byan aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL).The resulting yellow mixture was extracted with dichloromethane (3×15mL). The organic extracts were combined and then dried over anhydroussodium sulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a yellow oil. The product was purified bypreparatory HPLC on a Coulter Ultrasphere ODS column (5 μm, 250×10 mm,flow rate 3.5 mL/min, Solvent A: water, Solvent B: methanol, UVdetection at 350 nm) using an injection volume of 500 μL DMSO and agradient elution of 92-100% B over 30 min. The peak eluting at 21-29 minwas collected and concentrated to give enol CDL-II-466 as a light yellowsolid (15.0 mg, 67%).

R_(f) 0.55 (3:7 ethyl acetate-hexanes); ¹H NMR (600 MHz, CD₂Cl₂) δ 16.05(s, 1H, enol-OH), 7.52-7.26 (m, 10H, ArH), 6.66 (s, 1H, pyr-H), 5.57 (d,1H, J=12.7 Hz, OCHH′Ph), 5.43 (d, J=12.7 Hz, 1H, OCHH′Ph), 5.33-5.28 (m,2H, OCH₂Ph), 3.99 (d, 2H, J=10.5 Hz, CHN(CH₃)₂), 3.04-3.00 (m, 1H,CHCH₂CHCHN(CH₃)₂), 2.84 (dd, 1H, J=16.1, 4.9 Hz, CHH′CHCH₂CHCHN(CH₃)₂),2.74 (dd, 1H, J=16.1, 16.1 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.53 (dd, 1H,J=10.5, 3.9 Hz, CHCHN(CH₃)₂), 2.51-2.43 (m, 10H, N(CH₃)₂, Ar—CH₃,CHH′CHCHN(CH₃)₂), 2.07 (d, 1H, J=14.2 Hz, CHH′CHCHN(CH₃)₂), 0.82 (s, 9H,TBS), 0.22 (s, 3H, TBS), 0.10 (s, 3H, TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ187.9, 185.2, 182.5, 178.8, 167.9, 161.9, 161.8, 154.8, 137.9, 135.6,129.1, 129.0, 129.0, 128.7, 127.9, 127.9, 116.4, 111.6, 108.6, 107.5,82.0, 73.0, 68.1, 61.7, 46.9, 42.0, 39.2, 28.6, 26.1, 24.6, 23.0, 19.3,−2.4, −3.5; FTIR (neat film), cm⁻¹ 2939 (m), 2857 (w), 1720 (s, C═O),1593 (s), 1510 (s), 1469 (m), 1449 (m), 1326 (s), 1254 (m), 1187 (w),1157 (m), 1090 (m), 1064 (m), 1007 (m); HRMS (ES) m/z calcd for(C₄₁H₄₇N₃O₇Si+H)⁺ 722.3262. found 722.3261.

Pyridone Sancycline Analog CDL-II-460:

Palladium hydroxide on carbon (20 wt. % Pd, wet, water max. 50%, 10 mg,0.0094 mmol, 0.7 equiv) was added to a solution of the Michael-Dieckmannaddition product CDL-II-466 (10 mg, 0.014 mmol, 1.0 equiv) indioxane-methanol (1:1, 10 mL) at 23° C. An atmosphere of hydrogen gaswas introduced by briefly evacuating the flask, then flushing with purehydrogen (1 atm). The resulting mixture was stirred at 23° C. for 2 h.The color turned green after 5 min and then gradually to yellow withinthe reaction time. The mixture was filtered through a plug of cotton andthen concentrated to a yellow oil. Aqueous hydrochloric acid (37%, 100μL) was added to a solution of the residue in methanol (10 mL) at 23° C.The reaction was monitored by analytical HPLC on a Coulter UltrasphereODS column (5 μm, 250×4.6 mm, flow rate 1 ml/min, Solvent A: 0.1% TFA inwater, Solvent B: 0.1% TFA in acetonitrile, UV detection at 395 nm) witha gradient elution of 10-100% B over 15 min. The peak at 7.0 minindicated the desired product. After stirring for 3 h at 23° C. thedeprotection was complete and the mixture was concentrated to a yellowoil. The crude mixture was purified by preparatory HPLC on a PhenomenexPolymerx DVB column (10 μm, 250×10 mm, flow rate 4 ml/min, Solvent A:0.01 N aqueous hydrochloric acid, Solvent B: acetonitrile, UV detectionat 365 nm) using an injection volume of 500 μL methanol containingoxalic acid monohydrate (30 mg) and a linear gradient of 0-20% B over 40min. The peak eluting at 20-29 min was collected and concentrated togive the hydrochloride of CDL-II-460 as a yellow powder (4.8 mg, 74%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 6.37 (s, 1H, ArH), 4.06 (s, 1H,CHN(CH₃)₂), 3.05-2.95 (m, 8H, N(CH₃)₂, CHCHN(CH₃)₂, CHCH₂CHCHN(CH₃)₂),2.79 (dd, 1H, J=16.1, 3.9 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.55 (dd, 1H,J=16.1, 16.1 Hz, CHH′CHCH₂CHCHN(CH₃)₂)), 2.40 (s, 3H, Ar—CH₃), 2.18 (br.d, 1H, J=12.7 Hz, CHH′CHCHN(CH₃)₂), 1.59 (ddd, 1H, J=12.7, 12.7, 12.7Hz, CHH′CHCHN(CH₃)₂); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 187.3, 183.5, 177.8,172.1, 160.6, 159.8, 153.3, 115.3, 107.2, 106.9, 95.6, 74.2, 68.4, 41.5,35.7, 34.5, 33.9, 31.0, 19.2; HRMS (ES) m/z calcd for(C₂₁H₂₃N₃O₇+H)⁺430.1614. found 430.1607.

Example 5—Synthesis of Pyridine Sancycline Analog(7-Aza-10-Deoxysancycline)

A solution of 2-methyl-nicotinic acid ethyl ester JDB1-67-SM (0.589 g,3.56 mmol, 1.0 equiv), aqueous sodium hydroxide (1.0 M, 3.9 mL, 3.9mmol, 1.1 equiv), and ethanol (5 mL) was heated at reflux for 18 h. Thereaction mixture was allowed to cool to 23° C., and was concentrated,affording the carboxylate salt (710 mg) as a white solid. Oxalylchloride (357 μL, 4.09 mmol, 1.15 equiv) was added to a mixture of thecarboxylate salt in dichloromethane (20 mL) at 23° C. Vigorous gasevolution was observed upon addition. The reaction mixture was stirredat 23° C. for 30 min, then N,N-dimethylformamide (20 μL) was added.After stirring for an additional 30 min at 23° C., phenol (837 mg, 8.90mmol, 2.5 equiv), pyridine (864 μL, 10.7 mmol, 3.0 equiv), anddimethylaminopyridine (3 mg) were added in sequence. The resultingsolution was stirred for 90 min at 23° C., whereupon an aqueouspotassium phosphate buffer solution (pH 7.05, 0.2 M, 5.0 mL) was added.The resulting mixture was partitioned between water (30 mL) and ethylacetate (50 mL). The aqueous phase was extracted with an additional50-mL portion of ethyl acetate. The organic layers were combined andwashed with an aqueous sodium hydroxide solution (50 mL, 1M), brine (50mL), and then dried over anhydrous sodium sulfate. The dried solutionwas decanted and concentrated, affording a colorless oil (900 mg). Theproduct was purified by flash column chromatography (25:75 ethylacetate-hexanes), providing the ester JDB1-67 as a colorless oil (500mg, 66%).

R_(f) 0.15 (3:7 ethyl acetate-hexanes); ¹H NMR (300 MHz, CDCl₃) δ 8.70(dd, 1H, J=1.7, 4.95 Hz, pyr-H), 8.44 (dd, 1H, J=1.7, 7.8 Hz, pyr-H),7.48-7.43 (m, 2H, ArH), 7.33-7.20 (m, 4H, ArH, pyr-H), 2.93 (s, 1H,CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 160.8, 152.4, 150.5, 138.9,129.5, 126.1, 124.5, 121.6, 121.0, 25.0; FTIR (neat film), cm⁻¹ 3406(m), 1948 (w), 1747 (s), 1578 (s), 1487 (s), 1435 (s), 1273 (s), 1237(s), 1191 (s), 1046 (s), 915 (m), 822 (m), 749 (s), 689 (s); HRMS (ES)m/z calcd for (C₁₃H₁₁NO₂+H)⁺ 214.0868. found 214.0866.

A solution of n-butyllithium in hexanes (1.47 M, 136 μL, 0.200 mmol,8.03 equiv) was added to a solution of diisopropylamine (26.5 μL, 0.202mmol, 8.05 equiv) in tetrahydrofuran (0.750 mL) at −78° C. The reactionmixture was briefly (10 min) transferred to an ice bath, with stirring,then was cooled to −78° C. Hexamethylphosphoramide (49.0 μL, 0.399 mmol,16.0 equiv) was added to the mixture prepared above at −78° C. Theresulting mixture was stirred for 5 minutes whereupon a colorlesssolution was formed. The resulting solution was added dropwise viacannula to a solution of the ester JDB1-67 (36.0 mg, 0.169 mmol, 6.79equiv), and the enone DRS6 (12.2 mg, 0.0249 mmol, 1.00 equiv) intetrahydrofuran (1 mL) at −95° C. dropwise via cannula. The light redmixture was allowed to warm to −50° C. over 50 min and was thenpartitioned between an aqueous potassium phosphate buffer solution (pH7.0, 0.2 M, 5.0 mL) and dichloromethane (25 mL). The organic phase wasseparated and the aqueous phase was further extracted withdichloromethane (3×15 mL). The organic phases were combined and driedover anhydrous sodium sulfate. The dried solution was decanted andconcentrated, affording a yellow solid. The product was purified bypreparatory HPLC on a Coulter Ultrasphere ODS column (10 250×10 mm, 3.5mL/min, Solvent A: water, Solvent B: methanol, UV detection at 350 nm)using an injection volume of 500 μL methanol and a linear gradientelution of 85-100% B over 30 min. The peak at 21-27 min was collectedand concentrated to give enol JDB1-87 as a white solid (11.0 mg, 72%).

R_(f) 0.07 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CD₂Cl₂) δ 15.21(s, 1H, enol), 8.63 (d, 1H, J=4.5 Hz, pyr-H), 8.19 (d, 1H, J=7.5 Hz,pyr-H), 7.54-7.43 (m, 5H, ArH), 7.34 (d, 1H, J=4.5, 7.5 Hz, pyr-H), 5.36(d, 1H, J=12.0 Hz, OCHH′Ph), 5.33 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.03 (d,1H, J=10.7 Hz, CHN(CH₃)₂), 3.36-3.31 (m, 1H, CHCH₂CHCHN(CH₃)₂), 3.23(dd, 1H, J=16.3, 5.6 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.99 (dd, 1H, J=16.3,16.3 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.63 (ddd, 1H, J=1.6, 4.4, 10.7 Hz,CHCHN(CH₃)₂), 2.54-2.48 (m, 7H, N(CH₃)₂, CHH′CHCHN(CH₃)₂), 2.19 (dd, 1H,J=1.6, 14.5 Hz, CHH′CHCHN(CH₃)₂), 0.87 (s, 9H, TBS), 0.26 (s, 3H, TBS),0.13 (s, 3H, TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ 187.7, 183.5, 182.6,182.2, 167.9, 161.2, 153.4, 137.6, 134.1, 129.2, 129.1, 129.1, 126.8,123.0, 108.7, 106.9, 82.2, 73.0, 61.8, 47.0, 42.1, 41.4, 30.1, 28.4,26.1, 23.2, 19.3, −2.4, −3.5; HRMS (ES) m/z calcd for (C₃₃H₃₉N₃O₆Si+H)⁺602.2686. found 602.2686.

Pd black (3.0 mg, 0.028 mmol, 2.6 equiv) was added in one portion to asolution of the enol JDB1-87 (6.5 mg, 0.011 mmol, 1.0 equiv) indioxane-methanol (7:2, 9.0 mL) at 23° C. An atmosphere of hydrogen wasintroduced by briefly evacuating the flask, then flushing with purehydrogen (1 atm). The green mixture was stirred for 7 hr, and thenfiltered through a plug of cotton. The filtrate was concentrated,providing the carboxamide as a yellow oil (7.0 mg). Aqueous hydrofluoricacid (48%, 0.5 mL) was added to a polypropylene reaction vesselcontaining a solution of the carboxamide in acetonitrile (4.5 mL) at 23°C. The reaction mixture was heated to 35° C. and was stirred at thattemperature for 27 hr. The excess hydrofluoric acid was quenched withmethoxytrimethylsilane (3.5 mL, 25 mmol). The reaction mixture wasconcentrated, affording a yellow solid. The product was purified bypreparatory HPLC on a Phenomenex Polymerx DVB column (10 μm, 250×10 mm,4 mL/min, Solvent A: 0.5% trifluoroacetic acid in water, Solvent B: 0.5%trifluoroacetic acid in methanol-acetonitrile (1:1), UV detection at 350nm) using an injection volume of 500 μL methanol and a linear gradientof 0-20% B over 40 min. The peak at 35-45 min was collected andconcentrated to give a yellow oil. The oil was dissolved in 1 mLmethanol, treated with concentrated hydrochloric acid (20 μL), and thenconcentrated to give the hydrochloride of JDB1-109 as a yellow powder(3.7 mg, 86%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 8.79-8.77 (m, 2H, pyr-H) 7.91(dd, 1H, J=6.8, 6.8 Hz, pyr-H), 4.12 (s, 1H, CHN(CH₃)₂), 3.41-3.22 (m,2H, CHH′CHCH₂CHCHN(CH₃)₂, CHCH₂CHCHN(CH₃)₂), 3.11-3.00 (m, 8H,CHH′CHCH₂CHCHN(CH₃)₂, CHCHN(CH₃)₂, N(CH₃)₂), 2.34 (ddd, 1H, J=12.9, 4.4,2.4 Hz, CHH′CHCHN(CH₃)₂), 1.77 (ddd, 1H, J=12.9, 12.9, 12.9 Hz,CHH′CHCHN(CH₃)₂); HRMS (ES) m/z calcd for (C₂₀H₂₁N₃O₆+H)⁺ 400.1508.found 400.1504.

Example 6—Synthesis of 10-Deoxysancycline

N,N-dimethylformamide (20 μL) was added was added to a solution of thecarboxylic acid JDB1-113-SM (500 mg, 3.67 mmol, 1.0 equiv) and oxalylchloride (367 μl, 4.22 mmol, 1.15 equiv) in dichloromethane (20 mL) at23° C. Vigorous gas evolution was observed. After stirring for 80 min at23° C., phenol (863 mg, 9.18 mmol, 2.5 equiv), pyridine (890 μL, 11.0mmol, 3.0 equiv), and dimethylaminopyridine (3 mg) were added insequence. The resulting solution was stirred for 90 min at 23° C.,whereupon an aqueous potassium phosphate buffer solution (pH 7.05, 0.2M, 5.0 mL) was added. The resulting mixture was partitioned betweenwater (30 mL) and ethyl acetate (50 mL). The aqueous phase was extractedwith an additional 50-mL portion of ethyl acetate. The organic layerswere combined and washed with an aqueous sodium hydroxide solution (50mL, 1M), brine (50 mL), and then dried over anhydrous sodium sulfate.The dried solution was decanted and concentrated, affording a colorlessoil (850 mg). The product was purified by flash column chromatography(25:75 ethyl acetate-hexanes), providing the ester JDB1-113 as acolorless oil (774 mg, 99%).

R_(f) 0.43 (3:7 ethyl acetate-hexanes); ¹H NMR (300 MHz, CDCl₃) δ 8.18(d, 1H, J=8.1 Hz, ArH), 7.49-7.20 (m, 8H, ArH, OArH), 2.69 (s, 3H,ArCH₃); ¹³C NMR (100 MHz, CDCl₃) δ 165.8, 150.9, 141.3, 132.7, 132.0,131.2, 129.5, 128.5, 125.9, 125.8, 121.8, 22.0; FTIR (neat film), cm⁻¹3046 (w), 2923 (w), 1739 (s), 1594 (m), 1487 (m), 1287 (m), 1241 (s),1189 (s), 1159 (m), 1041 (s), 733 (s); HRMS (ES) m/z calcd for(C₁₄H₁₂O₂+NH₄)⁺ 230.1181. found 230.1187.

A solution of n-butyllithium in hexanes (1.47 M, 38.0 μL, 0.0565 mmol,8.26 equiv) was added to a solution of diisopropylamine (7.4 μL, 0.057mmol, 8.3 equiv) in tetrahydrofuran (0.50 mL) at −78° C. The reactionmixture was briefly (10 min) transferred to an ice bath, with stirring,then was cooled to −78° C. Hexamethylphosphoramide (13.9 μL, 0.113 mmol,16.5 equiv) was added to the mixture prepared above at −78° C. Theresulting mixture was stirred for 5 minutes whereupon a colorlesssolution was formed. The resulting solution was added dropwise viacannula to a solution of the ester JDB1-113 (10.0 mg, 0.0471 mmol, 6.88equiv), and the enone DRS6 (3.3 mg, 0.00684 mmol, 1.00 equiv) intetrahydrofuran (0.50 mL) at −95° C. dropwise via cannula. The light redmixture was allowed to warm to −70° C. over 30 min and was thenpartitioned between an aqueous potassium phosphate buffer solution (pH7.0, 0.2 M, 5.0 mL) and dichloromethane (20 mL). The organic phase wasseparated and the aqueous phase was further extracted with an additional20-mL portion of dichloromethane. The organic phases were combined anddried over anhydrous sodium sulfate. The dried solution was decanted andconcentrated, affording a yellow solid. The product was purified bypreparatory HPLC on a Coulter Ultrasphere ODS column (10 250×10 mm, 3.5mL/min, Solvent A: water, Solvent B: methanol, UV detection at 350 nm)using an injection volume of 500 μL methanol and a linear gradientelution of 85-100% B over 30 min. The peak at 25-30 min was collectedand concentrated to give enol JDB1-87 as a white solid (3.5 mg, 85%).

R_(f) 0.46 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CD₂Cl₂) δ 15.53(s, 1H, enol), 7.94 (d, 1H, J=7.9 Hz, ArH), 7.54-7.28 (m, 8H, ArH,OCH₂ArH), 5.37-5.34 (m, 2H, OCH₂Ph), 4.05 (d, 1H, J=10.7 Hz, CHN(CH₃)₂),3.24-3.18 (m, 1H, CHCH₂CHCHN(CH₃)₂), 2.99 (dd, 1H, J=15.5, 5.6 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.88 (dd, 1H, J=15.5, 15.5 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.61 (dd, 1H, J=4.4, 10.7 Hz, CHCHN(CH₃)₂),2.54-2.44 (m, 7H, N(CH₃)₂, CHH′CHCHN(CH₃)₂), 2.14 (d, 1H, J=14.3 Hz,CHH′CHCHN(CH₃)₂), 0.86 (s, 9H, TBS), 0.25 (s, 3H, TBS), 0.12 (s, 3H,TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ 187.8, 183.0, 182.8, 182.4, 167.7,141.7, 135.4, 133.4, 130.9, 129.0, 128.9, 128.9, 128.1, 127.5, 126.5,108.5, 106.8, 82.1, 72.8, 61.5, 58.5, 46.9, 41.9, 38.6, 29.0, 25.9,23.1, 19.1, −2.6, −3.7; HRMS (ES) m/z calcd for (C₃₄H₄₀N₃O₆Si+H)⁺601.2734. found 601.2730.

Hydrofluoric acid (1.1 mL, 48% aqueous) was added to a polypropylenereaction vessel containing a solution of the enol JDB1-114 (15.1 mg,0.0251 mmol, 1.0 equiv) in acetonitrile (10 mL) at 23° C. The resultingmixture was stirred vigorously at 23° C. for 12 hr, then was poured intowater (50 mL) containing K₂HPO₄ (4.7 g). The resulting mixture wasextracted with ethyl acetate (3×25 mL). The organic phases were combinedand dried over anhydrous sodium sulfate. The dried solution was filteredand the filtrate was concentrated, furnishing the intermediate alcoholas a yellow solid (12.2 mg, 99%). Pd black was added in one portion to asolution of the residue in methanol-dioxane (1:1, 3.0 mL). An atmosphereof hydrogen was introduced by briefly evacuating the flask, thenflushing with pure hydrogen (1 atm). The mixture was stirred at 23° C.for 20 min. Within 5 min, the color changed from light yellow to green.The reaction mixture was filtered through a plug of cotton. The filtratewas concentrated to a yellow solid (13 mg). The product was purified bypreparatory HPLC on a Phenomenex Polymerx DVB column (10 μm, 250×10 mm,flow rate 5 mL/min, Solvent A: 0.01 N HCl, Solvent B: acetonitrile, UVdetection at 350 nm) using an injection volume of 450 μL methanolcontaining oxalic acid monohydrate (10 mg) in two injections and alinear gradient elution of 5-50% B in 30 min. The peak eluting at 16-22min was collected and concentrated to give 10-deoxysancyclinehydrochloride (JDB1-130.HCl) as a white powder (9.1 mg, 91%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 7.96 (d, 1H, J=7.3 Hz, ArH)7.51 (dd, 1H, J=7.3, 7.3 Hz, ArH), 7.39 (dd, 1H, J=7.3, 7.3 Hz, ArH),7.30 (d, 1H, J=7.3 Hz, ArH), 4.04 (s, 1H, CHN(CH₃)₂), 3.31-2.99 (m, 8H,CHCH₂CHCHN(CH₃)₂, CHCHN(CH₃)₂, N(CH₃)₂), 2.87 (dd, 1H, J=15.4, 4.3 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.61 (dd, 1H, J=15.4, 15.4 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.21 (ddd, J=12.8, 5.0, 2.5 Hz, CHH′CHCHN(CH₃)₂),1.66 (ddd, 1H, J=12.8, 12.8, 12.8 Hz, CHH′CHCHN(CH₃)₂).

Example 7—A Convergent, Enantioselective Synthetic Route to StructurallyDiverse 6-Deoxytetracycline Antibiotics

Among tetracyclines, semi-synthetic approaches have led to the discoveryof the 6-deoxytetracyclines doxycycline (2 in FIG. 15A) and minocycline(3 in FIG. 15A), clinically the most important agents in the class.6-Deoxytetracyclines exhibit considerably improved chemical stability ascompared to their 6-hydroxy counterparts and show equal or greaterpotencies in antibacterial assays (Stephens et al., J. Am. Chem. Soc.85, 2643 (1963); M. Nelson, W. Hillen, R. A. Greenwald, Eds.,Tetracyclines in Biology, Chemistry and Medicine (Birkhauser Verlag,Boston, 2001); each of which is incorporated herein by reference). It isevident that at present neither semi-synthesis nor modified biosynthesisis capable of addressing the great majority of novel structures that achemist might wish to explore in pursuit of a lead structure liketetracycline; structures such as the D-ring heterocyclic analogs 4 and 5in FIG. 15A, or new ring systems such as the pentacycline 6 (FIG. 15A)are exemplary. Absent a viable laboratory synthetic pathway, thesestructures and the regions of complex chemical space they represent mustbe ceded in the search for new antibiotics. Here, we report a short andefficient route for the synthesis of enantiomerically pure members ofthe 6-deoxytetracyclines from benzoic acid. The route we describe allowsfor the synthesis of 6-deoxytetracyclines (both with or without anhydroxyl group at C5) by a notably late-stage coupling reaction of theAB precursors 7 or 8 (FIG. 15B) with a variety of different D-ringprecursors, and has provided compounds such as doxycycline (2 in FIG.15A), the heterocyclic analogs 4 and 5 (FIG. 15A), the pentacycline 6(FIG. 15A), as well as other 6-deoxytetracycline analogs.

The strategic advantage of a synthetic approach involving a late-stageC-ring construction (AB+D→ABCD, FIG. 15B) is that much of the polarfunctionality known to play a role in the binding of tetracyclines tothe bacterial ribosome lies within the AB fragment (D. E. Brodersen etal., Cell 103, 1143 (2000); M. Pioletti et al., EMBO J. 20, 1829 (2001);each of which is incorporated herein by reference), while enormousstructural variation on or near the D-ring is not only permissible, buthas been cited as a means to overcome bacterial resistance. The advancedclinical candidate tigecycline (P.-E. Sum, P. Petersen, Bioorg. Med.Chem. Lett. 9, 1459 (1999); incorporated herein by reference), aminocycline derivative with a D-ring substituent, is exemplary, and isreported to be one of the most promising new antibiotics underevaluation by the FDA (K. Bush, M. Macielag, M. Weidner-Wells, Curr.Opin. Microbiol. 7, 466 (2004); incorporated herein by reference).Classically, approaches to the synthesis of the tetracycline antibioticshave proceeded by stepwise assembly of the ABCD ring system and beginwith D or CD precursors, as exemplified by the Woodward synthesis of(±)-6-deoxy-6-demethyltetracycline (sancycline, 25 steps, ˜0.002% yield)(J. J. Korst et al., J. Am. Chem. Soc. 90, 439 (1968); incorporatedherein by reference), the Shemyakin synthesis of(±)-12a-deoxy-5a,6-anhydrotetracycline (A. I. Gurevich et al.,Tetrahedron Lett. 8, 131 (1967); incorporated herein by reference), andthe Muxfeldt synthesis of (±)-5-oxytetracycline (terramycin, 22 steps,0.06% yield) (H. Muxfeldt et al., J. Am. Chem. Soc. 101, 689 (1979);incorporated herein by reference). Only one published synthesis of(−)-tetracycline itself has appeared, this from D-glucosamine (an A-ringprecursor, 34 steps, 0.002% yield) (K. Tatsuta et al., Chem. Lett. 646(2000); incorporated herein by reference), while the most efficientconstruction of the tetracycline ring system thus far is undoubtedly thesynthesis of (±)-12a-deoxytetracycline by the Stork laboratory (16steps, 18-25% yield) (G. Stork et al., J. Am. Chem. Soc. 118, 5304(1996); incorporated herein by reference). The latter research served toidentify C12a oxygenation as perhaps the greatest challenge intetracycline synthesis (it could not be achieved with12a-deoxytetracycline as substrate), a conclusion supported by theresults of prior synthetic efforts (J. J. Korst et al., J. Am. Chem.Soc. 90, 439 (1968); A. I. Gurevich et al., Tetrahedron Lett. 8, 131(1967); H. Muxfeldt et al., J. Am. Chem. Soc. 101, 689 (1979); each ofwhich is incorporated herein by reference). The problem is significant,for C12a oxygenation appears to greatly enhance antimicrobial activity(W. Rogalski, in Handbook of Experimental Pharmacology, J. J. Hlavka, J.H. Boothe, Eds. (Springer-Verlag, New York, 1985), vol. 78, chap. 5;incorporated herein by reference). A key feature of the syntheticapproach to 6-deoxytetracyclines that we have developed is that itintroduces the C12a hydroxyl group in the first step of the sequence(FIG. 16) and uses the stereogenic center produced in that step toelaborate all others in the target molecule. To protect the vinylogouscarbamic acid function of the A-ring we used the 5-benzyloxyisoxazolegroup developed by Stork and Haggedorn for that purpose (G. Stork, A. A.Hagedorn, III, J. Am. Chem. Soc. 100, 3609 (1978); incorporated hereinby reference), an innovation that proved critically enabling in thepresent work, while the dimethylamino group of the A-ring wasincorporated without modification.

Our synthesis of 6-deoxytetracyclines was initiated by whole-cell,microbial dihydroxylation of benzoic acid with a mutant strain ofAlcaligenes eutrophus (A. M. Reiner, G. D. Hegeman, Biochemistry 10,2530 (1971); A. G. Myers et al., Org. Lett. 3, 2923 (2001); each ofwhich is incorporated herein by reference), producing the diol 9 (FIG.16) with >95% ee in 79% yield (90-g batch, ˜13 g/L, FIG. 16).Hydroxyl-directed epoxidation of the microcrystalline product (9,m-CPBA, EtOAc) provided the α-oriented epoxide 10 (FIG. 16) in 83%yield; esterification of this product (trimethylsilyldiazomethane)followed by bis-silylation and concomitant epoxide isomerization in thepresence of tert-butyldimethylsilyl triflate (3 equiv.), afforded theepoxy ester 11 (FIG. 16) in 70% yield (A. G. Myers et al., Org. Lett. 3,2923 (2001); incorporated herein by reference). Separately,3-benzyloxy-5-dimethylaminomethylisoxazole, prepared on the mole-scaleby a simple four-step sequence from glyoxylic acid (D. M. Vyas, Y.Chiang, T. W. Doyle, Tetrahedron Lett. 25, 487 (1984); P. Pevarello, M.Varasi, Synth. Commun. 22, 1939 (1992); each of which is incorporatedherein by reference), was deprotonated at C4 with n-butyllithium, andthe resulting organolithium reagent (12 in FIG. 16) was then added tothe epoxy ester 11 (FIG. 16), forming the ketone 13 (73%) (FIG. 16). Ina noteworthy transformation, and a key step of the synthesis, exposureof the ketone 13 (FIG. 16) to lithium triflate (5 mol %) at 60° C.,followed by selective removal of the allylic silyl ether of therearranged product (TFA), afforded the tricyclic AB precursor 14 (FIG.16) in 62% yield after purification by flash column chromatography. Thetransformation of 13 to 14 (FIG. 16) is believed to involve initialS_(N)-prime opening of the allylic epoxide by the N,N-dimethylaminogroup followed by ylide formation and [2,3]-sigmatropic rearrangement, aprocess that is reminiscent of the Sommelet-Hauser rearrangement (S. H.Pine, Organic Reactions, 18, 403 (1970); incorporated herein byreference). Compound 14 (FIG. 16) possesses the requisite cisstereochemistry of the AB fusion as well as an α-orientedN,N-dimethylamino substituent (confirmed by X-ray crystallographicanalysis of a derivative), and serves as a common intermediate for thesynthesis of both the AB precursor enone 7 (4 steps, 49% yield, FIG. 16)and the AB precursor to 5-α-hydroxy-6-deoxytetracyclines, enone 8 (8steps, 56% yield, FIG. 16), as detailed in sequence below.

To synthesize the AB precursor enone 7 (FIG. 16), intermediate 14 wassubjected to reductive transposition (A. G. Myers, B. Zheng, TetrahedronLett. 37, 4841 (1996); incorporated herein by reference) in the presenceof triphenylphosphine, diethyl azodicarboxylate, ando-nitrobenzenesulfonyl hydrazide (added last, a procedural variant),affording the transposed cycloalkene 15 in 74% yield. Hydrolysis of thesilyl ether group within 15 (HCl, methanol), oxidation of the resultingallylic alcohol (IBX, DMSO) (M. Frigerio, M. Santagostino, TetrahedronLett. 35, 8019 (1994); incorporated herein by reference), and protectionof the remaining (tertiary) carbinol (TBSOTf, 2,6-lutidine) (E. J. Coreyet al., Tetrahedron Lett. 22, 3455 (1981); incorporated herein byreference) then provided the enone 7 (FIG. 16) in 66% yield (3 steps)after flash column chromatography. By a somewhat longer but slightlymore efficient sequence the intermediate 14 (FIG. 16) could also betransformed into the enone 8 (FIG. 16), the AB precursor to5-α-hydroxy-6-deoxytetracyclines. This sequence involved thetransformation of 14 (FIG. 16) into the phenylthio ether 16 (with netretention), diastereoselective sulfoxidation using a chiral oxidant (F.A. Davis et al., J. Org. Chem. 57, 7274 (1992); incorporated herein byreference) (99:1 selectivity), and Mislow-Evans rearrangement (E. N.Prilezhaeva, Russ. Chem. Rev. 70, 897 (2001); incorporated herein byreference), producing the allylic alcohol 17 in 66% yield (4 steps).High diastereoselectivity in the sulfoxidation step was essential, foronly one diastereomer (the major isomer under the conditions specified)underwent efficient thermal rearrangement. After protection of theallylic alcohol 17 (FIG. 16) using benzyl chloroformate, a sequencenearly identical to the final three steps of the synthesis of 7 (FIG.16) was employed to transform the resulting benzyl carbonate into theenone 8 (FIG. 16) in 85% yield (56% yield and 8 steps from 14).

6-Deoxytetracyclines were assembled with all requisite functionality andstereochemistry in a single operation. In this process the AB precursors7 or 8 (FIG. 16)) are coupled with a range of different carbanionicD-ring precursors in a Michael-Dieckmann reaction sequence (T.-L. Ho,Tandem Organic Reactions (Wiley, New York, 1992); incorporated herein byreference) that forms two carbon-carbon bonds and the C-ring of the6-deoxytetracyclines (FIGS. 15B, 17, and 18A to 18C). The process isperhaps best illustrated in detail by the 3-step synthesis of(−)-doxycycline from the AB precursor 8 (FIG. 17). Deprotonation of theD-ring precursor 18 (4.5 equiv, LDA, TMEDA, THF, −78° C.), synthesizedin 5 steps (42% yield) from anisic acid, followed by addition of theenone 8 (1 equiv, −78→0° C.), provided the tetracyclic coupling product19 (FIG. 17) in diastereomerically pure form in 79% yield afterpurification by rp-HPLC. Removal of the protective groups (2 steps, 90%yield) and purification (rp-HPLC) afforded (−)-doxycycline hydrochloride(18 steps, 8.3% yield from benzoic acid). A remarkable feature of theconvergent coupling reaction that produces the tetracyclic product 19(FIG. 17) is its stereoselectivity. Although in theory fourdiastereomeric products can be formed, largely one was produced,corresponding in configuration (5aR, 6R) to that of known biologicallyactive 6-deoxytetracyclines. A minor diastereomeric impurity, believedto be 6-epi-19 (FIG. 17), was also isolated in separate rp-HPLCfractions (<7% yield). Michael-Dieckmann cyclization sequences (T.-L.Ho, Tandem Organic Reactions (Wiley, New York, 1992); incorporatedherein reference) and condensations of o-toluate anions in particular(F. J. Leeper, J. Staunton, J.C.S. Chem. Comm., 406 (1978); F. M.Hauser, R. P. Rhee, J. Org. Chem. 43, 178, (1978); J. H. Dodd, S. M.Weinreb, Tetrahedron Lett. 20, 3593 (1979); each of which isincorporated herein by reference) are extensively precedented insynthesis, but we are unaware of any example exhibiting the high degreeof diastereoselectivity of the present case. Phenyl ester activation intoluate condensations is also precedented, though in a system that formsa fully aromatized cyclization product (White et al., J. Org. Chem. 51,1150 (1986); incorporated herein by reference). We observed that thepresence of the phenyl ester group of the D-ring precursor 18 (FIG. 17)was essential for successful cyclization to occur; anions derived fromsimple alkyl esters and phthalide-derived anions underwent Michaeladdition, but the resulting adducts did not cyclize. Perhaps even moreremarkable than the condensation that produces 19 (FIG. 17) is theparallel transformation of 18 with the enone 7 (FIG. 18A, entry 1),which forms (−)-6-deoxytetracycline in protected form with >20:1diastereoselectivity, in 81% yield after purification by rp-HPLC(diastereomerically pure; a minor diastereomer, epimeric at C6, was alsoisolated separately). It appears that additions to 7 and 8 proceedalmost exclusively by addition to the “top” face of each enone (asdrawn), producing C5a-stereochemistry corresponding to naturaltetracyclines, though why this should be the case is not obvious.

As the examples of entries 2-5 (FIGS. 18A to 18C) show, efficient andstereoselective condensations are not restricted to the o-toluate anionderived from the D-ring substrate 18 (FIG. 17); the novel D-ringheterocyclic analogs 4 and 5 (FIGS. 18A and 18B) were synthesized by arelated sequence from o-toluate anions of very different structures, aswas the pentacyline derivative 6 (FIG. 18C). In each case it wasnecessary to optimize the specific conditions for o-toluate aniongeneration and trapping. For entries 3-5 (FIGS. 18B to 18C) aniongeneration was best conducted in situ, in the presence of the enone 7,either by selective deprotonation (entry 3) or by lithium-halogenexchange (entries 4 and 5). A number of potentially competingnon-productive reaction sequences (e.g., enolization of 7) might haveoccurred during in situ anion generation; the observed efficiencies ofthe transformations are surprising in light of this. It is alsonoteworthy that in situ anion generation permits the use of o-toluateslacking an o-alkoxy substituent (entries 3 and 4), substrates known tobe problematic from prior studies (F. M. Hauser et al., Synthesis 72(1980); incorporated herein by reference). Finally, o-toluate anionformation by in situ or stepwise halogen-metal exchange (entries 4 and5) is unprecedented.

The efficiencies of the synthetic sequences have allowed for thepreparation of sufficient quantities of each tetracycline analog forantibacterial testing using standard serial-dilution techniques (5-20 mgamounts). Minimum inhibitory concentrations (MICs) are reported for eachanalog in whole-cell antimicrobial assays using five Gram-positive andfive Gram-negative organisms (FIGS. 18A to 18C). Thus far, thepentacycline derivative 6 (FIG. 18C) has shown the most promisingantibacterial properties, with activity equal to or greater thantetracycline in each of the Gram-positive strains examined, includingstrains with resistance to tetracycline, methicillin, and vancomycin.

Experimentals

General Procedures.

All reactions were performed in flame-dried round bottomed or modifiedSchlenk (Kjeldahl shape) flasks fitted with rubber septa under apositive pressure of argon, unless otherwise noted. Air- andmoisture-sensitive liquids and solutions were transferred via syringe orstainless steel cannula. Organic solutions were concentrated by rotaryevaporation at ˜25 Torr (house vacuum). Flash column chromatography wasperformed on silica gel (60 Å, standard grade) as described by Still etal. (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,2923-2925; incorporated herein by reference). Analytical thin-layerchromatography was performed using glass plates pre-coated with 0.25 mm230-400 mesh silica gel impregnated with a fluorescent indicator (254nm). Thin-layer chromatography plates were visualized by exposure toultraviolet light and/or exposure to ceric ammonium molybdate or anacidic solution of p-anisaldehyde followed by heating on a hot plate.

Materials.

Commercial reagents and solvents were used as received with thefollowing exceptions. Triethylamine, diisopropylamine,N,N,N′,N′-tetramethylethylene-diamine, DMPU, HMPA, andN,N-diisopropylethylamine were distilled from calcium hydride under anatmosphere of dinitrogen. Dichloromethane, methanol, tetrahydrofuran,acetonitrile, and toluene were purified by the method of Pangborn et al.(Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;Timmers, F. J. Organometallics 1996, 15, 1518-1520; incorporated hereinby reference).

Instrumentation.

Proton nuclear magnetic resonance (¹H NMR) spectra and carbon nuclearmagnetic resonance (¹³C NMR) spectra were recorded with VarianUnity/Inova 600 (600 MHz), Varian Unity/Inova 500 (500 MHz/125 MHz), orVarian Mercury 400 (400 MHz/100 MHz) NMR spectrometers. Chemical shiftsfor protons are reported in parts per million (δ scale) and arereferenced to residual protium in the NMR solvents (CHCl₃: δ 7.26,C₆D₅H: δ 7.15, D₂HCOD: δ 3.31, CDHCl₂: δ 5.32, (CD₂H)CD₃SO: δ 2.49).Chemical shifts for carbon are reported in parts per million (δ scale)and are referenced to the carbon resonances of the solvent (CDCl₃: δ77.0, C₆D₆: δ 128.0, CD₃OD: δ 44.9, CD₂Cl₂: δ 53.8, (CD₃)₂SO: δ 39.5).Data are represented as follows: chemical shift, multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad),integration, coupling constant in Hz, and assignment. Infrared (IR)absorbance spectra were obtained using a Perkin-Elmer 1600 FT-IRspectrophotometer referenced to a polystyrene standard. Data arerepresented as follows: frequency of the absorption (cm⁻¹), intensity ofthe absorption (s=strong, m=medium, w=weak, br=broad), and assignment(where appropriate). Optical rotations were determined using a JASCODIP-370 digital polarimeter equipped with a sodium lamp source.High-resolution mass spectra were obtained at the Harvard UniversityMass Spectrometry Facilities.

Synthesis of (−)-Doxycycline

Cyclization Step:

A solution of n-butyllithium in hexanes (1.55 M, 155 μL, 0.240 mmol, 5.1equiv) was added to a solution of N,N,N′,N′-tetramethylethylenediamine(39 μL, 0.26 mmol, 5.5 equiv) and diisopropylamine (34 μL, 0.25 mmol,5.1 equiv) in tetrahydrofuran (1 mL) at −78° C. The resulting mixturewas stirred vigorously at −78° C. for 30 min whereupon a solution of2-(phenoxycarbonyl)-3-ethylphenyl t-butyl carbonate (73.0 mg, 0.213mmol, 4.5 equiv) in tetrahydrofuran (1 mL) was added dropwise viacannula. The resulting deep-red mixture was stirred vigorously at −78°C. for 75 min, then a solution of enone 8 (30.0 mg, 0.0474 mmol, 1equiv) in tetrahydrofuran (1 mL) was added dropwise via cannula. Theresulting light-red mixture was allowed to warm slowly to 0° C. over 2h. The ice-cold product solution was then partitioned between aqueouspotassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) anddichloromethane (10 mL). The organic phase was separated and the aqueousphase was further extracted with two 10-mL portions of dichloromethane.The organic phases were combined and dried over anhydrous sodiumsulfate. The dried solution was filtered and the filtrate wasconcentrated, providing a yellow oil. The product was purified bypreparatory HPLC on a Coulter Ultrasphere ODS column [10 μm, 250×10 mm,UV detection at 350 nm, injection volume: 400 μL (methanol), isochraticelution with methanol-water (9:1), flow rate: 3.5 mL/min]. Fractionseluting at 36-42 min were collected and concentrated, affording thepentacyclic addition product depicted in diastereomerically pure form(33.0 mg, 79%, a light-yellow solid).

R_(f) 0.35 (1:4 ethyl acetate-hexanes); ¹H NMR (500 MHz, C₆D₆) δ 16.55(br s, 1H, enol), 7.26 (d, 2H, J=7.0 Hz, o-ArH), 7.14 (d, 2H, J=7.5 Hz,ArH), 6.85-7.05 (m, 6H, ArH), 6.66-6.74 (m, 2H, ArH), 6.51 (dd, 1H,J=9.0, 1.5 Hz, ArH), 5.73 (br d, 1H, J=4.0 Hz, BnOCO₂CH), 5.17 (d, 1H,J=12.5 Hz, OCHH′Ph), 5.03 (d, 1H, J=12.5 Hz, OCHH′Ph), 4.99 (d, 1H,J=12.5 Hz, OCHH′Ph′), 4.93 (d, 1H, J=12.5 Hz, OCHH′Ph′), 3.58 (d, 1H,J=11.5 Hz, CHCHN(CH₃)₂), 3.35 (dd, 1H, J=12.5, 4.0 Hz, CH₃CHCH), 2.99(d, 1H, J=11.5 Hz, CHCHN(CH₃)₂), 2.56 (dq, 1H, J=12.5, 7.0 Hz, CH₃CH),2.18 (s, 6H, N(CH₃)₂), 1.33 (s, 9H, C(CH₃)₃), 1.16 (d, 3H, J=7.0 Hz,CH₃CH), 1.11 (s, 9H, C(CH₃)₃), 0.61 (s, 3H, CH₃), 0.36 (s, 3H, CH₃); ¹³CNMR (100 MHz, CDCl₃) δ 189.7, 186.3, 180.9, 178.4, 167.9, 154.7, 152.1,150.8, 145.9, 136.1, 135.5, 133.9, 128.7, 128.6, 128.5, 127.3, 123.8,122.7, 122.6, 108.9, 105.5, 83.0, 82.9, 74.8, 72.4, 69.2, 60.8, 52.7,43.2, 38.4, 27.5, 26.6, 19.5, 16.3, −1.8, −2.7; FTIR (neat film), cm⁻¹2974 (w), 2933 (w), 2851 (w), 1760 (s, C═O), 1748 (s, C═O), 1723 (s,C═O), 1606 (m), 1513 (m), 1471 (m), 1370 (m). 1260 (s), 1232 (s), 1148(s); HRMS (ES) m/z calcd for (C₄₈H₅₆N₂O₁₂Si)⁺ 881.3681. found 881.3684.

Deprotection Step 1:

Concentrated aqueous hydrofluoric acid (48 wt %, 1.2 mL) was added to apolypropylene reaction vessel containing a solution of the purifiedpentacyclic addition product from the experiment above (33.0 mg, 0.0375mmol, 1 equiv) in acetonitrile (7.0 mL) at 23° C. The resulting mixturewas stirred vigorously at 23° C. for 60 h, then was poured into water(50 mL) containing dipotassium hydrogenphosphate (7.0 g). The resultingmixture was extracted with ethyl acetate (3×20 mL). The organic phaseswere combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, affording theproduct depicted as a yellow oil (25.0 mg, 100%). This product was usedin the next step without further purification.

R_(f) 0.05 (1:4 ethyl acetate-hexanes); ¹H NMR (600 MHz, C₆D₆, crude) δ14.86 (br s, 1H, enol), 11.95 (s, 1H, phenol), 7.23 (d, 2H, J=7.8 Hz,o-ArH), 7.14 (d, 2H, J=7.2 Hz, o-ArH), 6.94-7.02 (m, 6H, ArH), 6.86 (t,1H, J=8.4 Hz, ArH), 6.76 (d, 1H, J=8.4 Hz, ArH), 6.28 (d, 1H, J=7.8 Hz,ArH), 5.46 (dd, 1H, J=3.6, 3.0 Hz, BnOCO₂CH), 5.12 (d, 1H, J=12.0 Hz,OCHH′Ph), 5.04 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.92 (s, 2H, OCH₂Ph), 3.41(d, 1H, J=9.6 Hz, CHCHN(CH₃)₂), 2.82 (dd, 1H, J=9.6, 3.0 Hz,CHCHN(CH₃)₂), 2.65 (dd, 1H, J=13.2, 3.6 Hz, CH₃CHCH), 2.78 (dq, 1H,J=13.2, 7.2 Hz, CH₃CH), 2.05 (s, 6H, N(CH₃)₂), 1.04 (d, 3H, J=7.2 Hz,CH₃CH); ¹³C NMR (100 MHz, C₆D₆, crude) δ 193.4, 186.2, 181.3, 172.3,167.9, 163.3, 154.6, 145.8, 136.6, 135.8, 128.6, 128.4, 127.2, 116.8,116.0, 115.6, 107.6, 104.7, 76.8, 73.9, 72.5, 69.5, 60.3, 48.7, 43.0,41.8, 37.5, 15.3; FTIR (neat film), cm⁻¹ 3424 (m, OH), 3059, 3030, 2925,2857, 1744 (s, C═O), 1713 (s, C═O), 1614 (s), 1582 (s), 1455 (s), 1252(s); HRMS (ES) m/z calcd for (C₃₇H₃₄N₂O₁₀+H)⁺ 667.2292. found 667.2300.

Deprotection Step 2:

Palladium black (7.00 mg, 0.0657 mmol, 1.75 equiv) was added in oneportion to a solution of the product from the procedure above (25.0 mg,0.0375 mmol, 1 equiv) in tetrahydrofuran-methanol (1:1, 2.0 mL) at 23°C. An atmosphere of hydrogen was introduced by briefly evacuating theflask, then flushing with pure hydrogen (1 atm). The palladium catalystwas initially observed to be a fine dispersion, but aggregated intoclumps within 5 min. The yellow heterogeneous mixture was stirred at 23°C. for 2 h, then was filtered through a plug of cotton. The filtrate wasconcentrated, affording a yellow oil. The product was purified bypreparatory HPLC on a Phenomenex Polymerx DVB column (10 250×10 mm, UVdetection at 350 nm, Solvent A: methanol-0.005 N aq. HCl (1:4), SolventB: acetonitrile, injection volume: 400 μL (solvent A containing 10 mgoxalic acid), isochratic elution with 5% B for 2 min, then gradientelution with 5→50% B for 20 min, flow rate: 4.0 mL/min]. Fractionseluting at 12-17 min were collected and concentrated, affording(−)-doxycycline hydrochloride as a yellow powder (16.2 mg, 90%), whichwas identical with natural (−)-doxycycline hydrochloride [reverse-phaseHPLC (co-injection), ¹H NMR (including measurement of an admixture ofsynthetic and natural doxycycline), ¹³C NMR, [α]_(D), UV).

¹H NMR (600 MHz, CD₃OD, hydrochloride) δ 7.47 (t, 1H, J=8.4 Hz, ArH),6.93 (d, 1H, J=8.4 Hz, ArH), 6.83 (d, 1H, J=8.4 Hz, ArH), 4.40 (s, 1H,(CH₃)₂NCH), 3.53 (dd, 1H, J=12.0, 8.4 Hz, CHOH), 2.95 (s, 3H,N(CH₃)CH₃′), 2.88 (s, 3H, N(CH₃)CH₃′), 2.80 (d, 1H, J=12.0 Hz,CHCHN(CH₃)₂), 2.74 (dq, 1H, J=12.6, 6.6 Hz, CH₃CH), 2.58 (dd, 1H,J=12.6, 8.4 Hz, CH₃CHCH), 1.55 (d, 3H, J=6.6 Hz, CH₃CHCH); ¹³C NMR (100MHz, CD₃OD) δ 195.3, 188.2, 173.8, 172.1, 163.2, 149.0, 137.7, 117.1,116.9, 116.6, 108.4, 96.0, 74.5, 69.8, 66.9, 47.5, 43.4, 43.0, 41.9,40.0, 16.3; UV max (0.01 M methanolic HCl), nm 218, 267, 350;[α]_(D)=−109° (c=0.16 in 0.01 M methanolic HCl); HRMS (ES) m/z calcd for(C₂₂H₂₄N₂O₈+H)⁺ 445.1611. found 445.1603.

Literature values (The Merck Index: An Encyclopedia of Chemicals, Drugs,and Biologicals, 12^(th) ed. Budavari, S.; O'Neal, M. J.; Smith, A.;Heckelman, P. E.; Kinneary, J. F., Eds.; Merck & Co.: WhitehouseStation, N J, 1996; entry 3496.): UV max (0.01 M methanolic HCl), nm267, 351; [α]_(D)=−110° (c=1 in 0.01 M methanolic HCl).

Synthesis of (−)-6-Deoxytetracycline

Cyclization Step:

A solution of n-butyllithium in hexanes (1.65 M, 75 μL, 0.12 mmol, 3.9equiv) was added to a solution of diisopropylamine (17 μL, 0.12 mmol,3.9 equiv) and N,N,N′,N′-tetramethylethylenediamine (19 μL, 0.13 mmol,4.1 equiv) in tetrahydrofuran (1 mL) at −78° C. The resulting solutionwas stirred at −78° C. for 30 min whereupon a solution of2-(phenoxycarbonyl)-3-ethylphenyl t-butyl carbonate (31.8 mg, 0.093mmol, 3.0 equiv) in tetrahydrofuran (250 μL) was added dropwise viasyringe. The resulting deep-red mixture was stirred at −78° C. for 90min, then a solution of enone 7 (15.0 mg, 0.031 mmol, 1 equiv) intetrahydrofuran (250 μL) was added dropwise via syringe. The resultingdeep-red mixture was allowed to warm slowly to 0° C. over 3 h. Theice-cold product solution was then partitioned between aqueous potassiumphosphate buffer solution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15mL). The organic phase was separated and the aqueous phase was furtherextracted with two 15-mL portions of dichloromethane. The organic phaseswere combined and dried over anhydrous sodium sulfate. The driedsolution was filtered and the filtrate was concentrated, providing ayellow oil. The product was purified by preparatory HPLC on a CoulterUltrasphere ODS column [5 μm, 250×10 mm, UV detection at 350 nm,injection volume: 500 μL (methanol), isochratic elution withmethanol-water (89:11), flow rate: 3.5 mL/min]. Fractions eluting at39-60 min were collected and concentrated, affording the pentacyclicaddition product depicted in diastereomerically pure form (18.5 mg, 81%,a light-yellow foam).

R_(f) 0.37 (2:8 tetrahydrofuran-hexanes); ¹H NMR (500 MHz, CDCl₃) δ (s,1H, 16.24, enol-OH), 7.55-7.50 (m, 3H, ArH), 7.40-7.35 (m, 4H, ArH),7.10 (d, 1H, J=7.8 Hz, ArH), 5.39-5.34 (m, 2H, OCH₂Ph), 3.92 (d, 1H,J=10.7 Hz, CHN(CH₃)₂), 2.81-2.71 (m, 2H, CH₃CH, CH₃CHCH), 2.55 (dd, 1H,J=10.7, 5.7 Hz, CHCHN(CH₃)₂), 2.48 (s, 6H, N(CH₃)₂), 2.40 (d, 1H, J=14.7Hz, CHH′CHCHN(CH₃)₂), 2.31 (ddd, 1H, J=14.7, 9.3, 5.7, CHH′CHCHN(CH₃)₂),1.56 (s, 3H, CH₃), 1.55 (s, 9H, Boc), 0.84 (s, 9H, TBS), 0.27 (s, 3H,TBS), 0.13 (s, 3H, TBS); ¹³C NMR (125 MHz, CDCl₃) δ 187.4, 183.1, 182.8,181.6, 167.6, 151.7, 150.2, 147.4, 135.0, 134.0, 128.5, 128.5, 123.4,123.0, 122.4, 108.3, 107.4, 94.8, 83.9, 81.5, 72.5, 61.5, 46.4, 41.9,39.5, 34.9, 27.7, 26.0, 20.7, 19.0, 16.0, −2.6, −3.7; FTIR (neat film),cm⁻¹ 2923 (m), 2841 (m), 1759 (s, C═O), 1718 (s, C═O), 1605 (s), 1508(s), 1467 (m), 1456 (m), 1369 (m), 1277 (s), 1262 (m), 1231 (s), 1144(s), 1005 (w); HRMS (ES) m/z calcd for (C₄₀H₅₀N₂O₉Si+H)⁺ 731.3364. found731.3370.

Deprotection:

Concentrated aqueous hydrofluoric acid solution (48 wt %, 0.6 mL) wasadded to a polypropylene reaction vessel containing a solution of thepurified pentacyclic addition product from the experiment above (15.0mg, 0.0205 mmol, 1 equiv) in acetonitrile (3.5 mL) at 23° C. Thereaction mixture was stirred at 23° C. for 55 h, then was poured intowater (20 mL) containing dipotassium hydrogenphosphate (4.0 g). Theresulting mixture was extracted with ethyl acetate (4×20 mL). Theorganic phases were combined and dried over anhydrous sodium sulfate.The dried solution was filtered and the filtrate was concentrated,affording a light-yellow oil. The residue was dissolved inmethanol-tetrahydrofuran (1:1, 2 mL) and to the resulting solution wasadded palladium black (7.6 mg, 0.071 mmol, 3.5 equiv) in one portion. Anatmosphere of hydrogen gas was introduced by briefly evacuating theflask, then flushing with pure hydrogen (1 atm). The yellow mixture wasstirred at 23° C. for 2 h, then was filtered through a plug of cotton.The filtrate was concentrated, affording a yellow oil (10 mg). Theproduct was purified by preparatory HPLC on a Phenomenex Polymerx DVBcolumn [10 μm, 250×10 mm, UV detection at 365 nm, Solvent A:methanol-0.02 N HCl (1:4), Solvent B: acetonitrile, injection volume:400 μL (methanol containing 10 mg oxalic acid), isochratic elution with18% B for 15 min, then gradient elution with 18→60% B over 15 min, flowrate: 5 mL/min]. Fractions eluting at 17.5-22.5 min were collected andconcentrated, affording 6-deoxytetracycline hydrochloride as a yellowpowder (8.1 mg, 85%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 7.49 (t, 1H, J=7.8 Hz, ArH),6.95 (d, 1H, J=7.8 Hz, ArH), 6.84 (d, 1H, J=7.8 Hz, ArH), 4.09 (s, 1H,CHN(CH₃)₂), 3.03 (br s, 3H, N(CH₃)), 2.97 (br s, 3H, N(CH₃)), 2.90 (brd, 1H, J=12.7 Hz, CHCHN(CH₃)₂), 2.67 (ddd, 1H, J=12.7, 12.7, 5.2 Hz,CH₃CHCH), 2.61-2.56 (m, 1H, CH₃CH), 2.30 (ddd, 1H, J=13.7, 5.2, 2.9 Hz,CHH′CHCHN(CH₃)₂), 1.54 (ddd, 1H, J=13.7, 12.7, 12.7 Hz,CHH′CHCHN(CH₃)₂), 1.38 (d, 3H, J=6.8 Hz, CH₃CH); UV max (0.01 Mmethanolic HCl), nm 269, 353; [α]_(D)=−142° (c=0.20 in 0.01 M methanolicHCl); HRMS (ES) m/z calcd for (C₂₂H₂₄N₂O₇+H)⁺ 429.1662. found 429.1660.

Synthesis of a (−)-D-ring Pyridone Analog of Tetracycline

Cyclization Step:

A solution of n-butyllithium in hexanes (1.67 M, 80 μL, 0.13 mmol, 4.3equiv) was added to a solution of diisopropylamine (20 μL, 0.14 mmol,4.6 equiv) in tetrahydrofuran (2.5 mL) at −78° C. The resulting solutionwas allowed to warm to 0° C. over 15 min. N,N′-dimethylpropyleneurea (17μL, 0.14 mmol, 4.5 equiv) was added and the resulting solution wascooled to −78° C. A solution of phenyl2-(benzyloxy)-4,6-dimethylpyridine-3-carboxylate (31.0 mg, 0.0930 mmol,2.99 equiv) in tetrahydrofuran (250 μL) was then added via syringe tothe cooled reaction solution. The resulting yellow solution was stirredfor 5 min at −78° C., then a solution of enone 7 (15.0 mg, 0.0311 mmol,1 equiv) in tetrahydrofuran (250 μL) was added via syringe. Theresulting deep-red mixture was allowed to warm to 0° C. over 4 h. Aceticacid (40 μL) was added to the deep-red mixture at 0° C. The ice-coldproduct solution was then partitioned between aqueous potassiumphosphate buffer solution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15mL). The organic phase was separated and the aqueous phase was furtherextracted with two 15-mL portions of dichloromethane. The organicextracts were combined and then dried over anhydrous sodium sulfate. Thedried solution was filtered and the filtrate was concentrated, providinga yellow oil. The product was purified by preparatory HPLC on a CoulterUltrasphere ODS column [5 μm, 250×10 mm, UV detection at 350 nm, SolventA: water, Solvent B: methanol, injection volume: 500 μL DMSO, gradientelution with 92→100% B over 30 min, flow rate: 3.5 mL/min]. Fractionseluting at 21-29 min were collected and concentrated, affording thepentacyclic addition product depicted in diasteromerically pure form(15.0 mg, 67%, a light-yellow solid).

R_(f) 0.55 (3:7 ethyl acetate-hexanes); ¹H NMR (600 MHz, CD₂Cl₂) δ 16.05(s, 1H, enol-OH), 7.52-7.26 (m, 10H, ArH), 6.66 (s, 1H, pyr-H), 5.57 (d,1H, J=12.7 Hz, OCHH′Ph), 5.43 (d, J=12.7 Hz, 1H, OCHH′Ph), 5.33-5.28 (m,2H, OCH₂Ph), 3.99 (d, 2H, J=10.5 Hz, CHN(CH₃)₂), 3.04-3.00 (m, 1H,CHCH₂CHCHN(CH₃)₂), 2.84 (dd, 1H, J=16.1, 4.9 Hz, CHH′CHCH₂CHCHN(CH₃)₂),2.74 (dd, 1H, J=16.1, 16.1 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.53 (dd, 1H,J=10.5, 3.9 Hz, CHCHN(CH₃)₂), 2.51-2.43 (m, 10H, N(CH₃)₂, Ar—CH₃,CHH′CHCHN(CH₃)₂), 2.07 (d, 1H, J=14.2 Hz, CHH′CHCHN(CH₃)₂), 0.82 (s, 9H,TBS), 0.22 (s, 3H, TBS), 0.10 (s, 3H, TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ187.9, 185.2, 182.5, 178.8, 167.9, 161.9, 161.8, 154.8, 137.9, 135.6,129.1, 129.0, 129.0, 128.7, 127.9, 127.9, 116.4, 111.6, 108.6, 107.5,82.0, 73.0, 68.1, 61.7, 46.9, 42.0, 39.2, 28.6, 26.1, 24.6, 23.0, 19.3,−2.4, −3.5; FTIR (neat film), cm⁻¹ 2939 (m), 2857 (w), 1720 (s, C═O),1593 (s), 1510 (s), 1469 (m), 1449 (m), 1326 (s), 1254 (m), 1187 (w),1157 (m), 1090 (m), 1064 (m), 1007 (m); HRMS (ES) m/z calcd for(C₄₁H₄₇N₃O₇Si+H)⁺ 722.3262. found 722.3261.

Deprotection:

Pearlman's catalyst (10 mg, 0.0094 mmol, 0.68 equiv) was added to asolution of the purified pentacyclic addition product from theexperiment above (10 mg, 0.014 mmol, 1 equiv) in dioxane-methanol (1:1,10 mL) at 23° C. An atmosphere of hydrogen gas was introduced by brieflyevacuating the flask, then flushing with pure hydrogen (1 atm). Thereaction mixture was observed to form a green color within 10 min. Afterstirring at 23° C. for 2 h, the reaction mixture was filtered through aplug of cotton and the filtrate was concentrated. The oily yellowresidue was dissolved in methanol (10 mL) and to the resulting solutionwas added concentrated aqueous hydrochloric acid solution (37 wt %, 100μL) at 23° C. The reaction mixture was stirred at 23° C. for 3 h, thenwas concentrated. The product was purified by preparatory HPLC on aPhenomenex Polymerx DVB column [10 μm, 250×10 mm, UV detection at 365nm, Solvent A: 0.01 N aqueous hydrochloric acid, Solvent B:acetonitrile, injection volume: 500 μL (methanol containing 30 mg oxalicacid), linear gradient with 0→20% B over 40 min, flow rate: 4 ml/min].Fractions eluting at 20-29 min were collected and concentrated,affording the D-ring pyridone hydrochloride as a yellow powder (4.8 mg,74%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 6.37 (s, 1H, ArH), 4.06 (s, 1H,CHN(CH₃)₂), 3.05-2.95 (m, 8H, N(CH₃)₂, CHCHN(CH₃)₂, CHCH₂CHCHN(CH₃)₂),2.79 (dd, 1H, J=16.1, 3.9 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.55 (dd, 1H,J=16.1, 16.1 Hz, CHH′CHCH₂CHCHN(CH₃)₂)), 2.40 (s, 3H, Ar—CH₃), 2.18 (br.D, 1H, J=12.7 Hz, CHH′CHCHN(CH₃)₂), 1.59 (ddd, 1H, J=12.7, 12.7, 12.7Hz, CHH′CHCHN(CH₃)₂); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 187.3, 183.5, 177.8,172.1, 160.6, 159.8, 153.3, 115.3, 107.2, 106.9, 95.6, 74.2, 68.4, 41.5,35.7, 34.5, 33.9, 31.0, 19.2; UV max (0.01 M methanolic HCl), nm 267,370; [α]_(D)=−146° (c=0.43 in 0.01 M methanolic HCl); HRMS (ES) m/zcalcd for (C₂₁H₂₃N₃O₇+H)⁺ 430.1614. found 430.1607.

Synthesis of a (−)-Pentacycline

Cyclization Step:

A solution of n-butyllithium in hexanes (2.65 M, 107 μL, 0.284 mmol,4.03 equiv) was added to a solution of phenyl3-(bromomethyl)-1-methoxynaphthalene-2-carboxylate (105 mg, 0.283 mmol,4.02 equiv) and enone 7 (34.0 mg, 0.0705 mmol, 1 equiv) intetrahydrofuran (2.80 mL) at −100° C. The resulting light-red reactionmixture was allowed to warm to 0° C. over 70 min. The ice-cold productsolution was then partitioned between aqueous potassium phosphate buffersolution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15 mL). The organicphase was separated and the aqueous phase was further extracted with two15-mL portions of dichloromethane. The organic phases were combined anddried over anhydrous sodium sulfate. The dried solution was filtered,and the filtrate was concentrated, affording a yellow solid. The productwas purified by preparatory HPLC on a Coulter Ultrasphere ODS column [10μm, 250×10 mm, UV detection at 350 nm, Solvent A: water, Solvent B:methanol, two separate injections (750 μL each, acetonitrile),isochratic elution with 94% B for 20 min followed by a linear gradientelution with 94→100% B over 20 min, flow rate: 3.5 mL/min]. Fractionseluting at 24-38 min were collected and concentrated, affording thehexacyclic addition product in diastereomerically pure form (36.1 mg,75%, a white solid).

R_(f) 0.37 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CDCl₃) δ 16.25(s, 1H, enol-OH), 8.30 (d, 1H, J=8.3 Hz, ArH), 7.75 (d, 1H, J=7.8 Hz,ArH), 7.59-7.34 (m, 7H, ArH), 7.26 (s, 1H, ArH), 5.38 (s, 2H, OCH₂Ph),4.02 (s, 3H, OCH₃), 3.99 (d, 1H, J=10.7 Hz, CHN(CH₃)₂), 3.08-3.05 (m,2H, CHCH₂CHCHN(CH₃)₂, CHH′CHCH₂CHCHN(CH₃)₂), 2.95-2.90 (m, 1H,CHH′CHCH₂CHCHN(CH₃)₂), 2.58 (dd, 1H, J=10.7, 5.9 Hz, CHCHN(CH₃)₂), 2.51(s, 6H, N(CH₃)₂), 2.50-2.48 (m, 1H, CHH′CHCHN(CH₃)₂), 2.20-2.14 (m, 1H,CHH′CHCHN(CH₃)₂), 0.82 (s, 9H, TBS), 0.29 (s, 3H, TBS), 0.13 (s, 3H,TBS); ¹³C NMR (125 MHz, CDCl₃) δ 187.9, 184.1, 183.0, 182.0, 167.8,159.2, 137.5, 136.7, 135.3, 129.5, 128.8, 128.7, 128.5, 127.5, 126.4,124.2, 121.8, 119.5, 108.7, 108.7, 82.4, 72.8, 63.8, 61.6, 46.8, 42.1,40.7, 29.3, 26.2, 23.1, 19.3, −2.2, −3.5; FTIR (neat film), cm⁻¹ 2934(m), 2852 (m), 1718 (s, C═O), 1610 (s), 1513 (s), 1472 (m), 1452 (m),1369 (m), 1339 (w), 1293 (m), 1252 (m), 1190 (w), 1159 (m), 1067 (m),1026 (w), 1011 (w); HRMS (ES) m/z calcd for (C₃₉H₄₄N₂O₇Si+H)⁺ 681.2996.found 681.2985.

Deprotection:

Concentrated aqueous hydrofluoric acid solution (48 wt %, 1.0 mL) wasadded to a polypropylene reaction vessel containing a solution of thepurified hexacyclic addition product from the experiment above (24.0 mg,0.035, 1 equiv) in acetonitrile (9.0 mL) at 23° C. The reaction mixturewas stirred at 23° C. for 22 h, then was poured into water (50 mL)containing dipotassium hydrogenphosphate (12.0 g). The resulting mixturewas extracted with ethyl acetate (3×50 mL). The organic phases werecombined and dried over anhydrous sodium sulfate. The dried solution wasfiltered and the filtrate was concentrated, affording a yellow oil. Theresidue was dissolved in methanol-dioxane (1:1, 5 mL) and to theresulting solution was added palladium black (10.0 mg, 0.0940 mmol, 2.67equiv) in one portion. An atmosphere of hydrogen gas was introduced bybriefly evacuating the flask, then flushing with pure hydrogen (1 atm).The yellow mixture was stirred at 23° C. for 4 h, then was filteredthrough a plug of cotton. The filtrate was concentrated, affording ayellow oil. The residue was dissolved in dichloromethane (4.5 mL) and tothe resulting solution was added a solution of boron tribromide (1.0 Min dichloromethane, 0.5 mL, 14 equiv) at −78° C. The dark-red mixturewas stirred at −78° C. for 15 min, then at 23° C. for 3.5 h. Methanol(20 mL) was added and the resulting yellow solution was stirred at 23°C. for 1 h. The solution was concentrated, affording a yellow oil. Theproduct was purified by preparatory HPLC on a Phenomenex Polymerx DVBcolumn [7 μm, 150×21.2 mm, UV detection at 350 nm, Solvent A: 0.01 NHCl, Solvent B: acetonitrile, injection volume: 500 μL (methanolcontaining 10 mg oxalic acid), gradient elution with 25→50% B over 60min, flow rate: 6 mL/min]. Fractions eluting at 30-35 min were collectedand concentrated, affording the pentacycline hydrochloride as a yellowpowder (13.1 mg, 74%).

¹H NMR (600 MHz, CD₃OD, hydrochloride) δ 8.36 (d, 1H, J=7.7 Hz, ArH),7.74 (d, 1H, J=7.7 Hz, ArH), 7.64 (dd, 1H, J=7.7, 7.7 Hz, ArH), 7.50(dd, 1H, J=7.7, 7.7 Hz, ArH), 7.1 (s, 1H, ArH), 4.10 (s, 1H, CHN(CH₃)₂),3.13-2.97 (m, 9H, N(CH₃)₂, CHCHN(CH₃)₂, CHCH₂CHCHN(CH₃)₂,CHH′CHCH₂CHCHN(CH₃)₂), 2.67 (dd, 1H, J=14.3, 14.3 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.22 (ddd, 1H, J=13.6, 4.9, 2.9 Hz,CHH′CHCHN(CH₃)₂), 1.64 (ddd, 1H, J=13.6, 13.6, 13.6 Hz,CHH′CHCHN(CH₃)₂); UV max (0.01 M methanolic HCl), nm 268, 345, 402;[α]_(D)=−113° (c=0.18 in 0.01 M methanolic HCl); HRMS (ES) m/z calcd for(C₂₅H₂₄N₂O₇+H)⁺ 465.1662. found 465.1656.

Synthesis of (−)-7-Aza-10-Deoxysancycline

Cyclization Step:

A solution of n-butyllithium in hexanes (2.65 M, 33.0 μL, 0.0945 mmol,5.00 equiv) was added to a solution of diisopropylamine (13.2 μL, 0.0945mmol, 5.00 equiv) in tetrahydrofuran (0.750 mL) at −78° C. The resultingsolution was briefly warmed in an ice bath (10 min), then was cooled to−78° C. Hexamethylphosphoramide (33.0 μL, 0.189 mmol, 10.0 equiv) wasadded, producing a colorless solution, and this solution was thentransferred (cold) dropwise via cannula to a solution containing phenyl2-methylpyridine-3-carboxylate (16.0 mg, 0.0755 mmol, 4.00 equiv) andenone 7 (9.1 mg, 0.019 mmol, 1 equiv) in tetrahydrofuran (0.750 mL) at−95° C., forming a light-red mixture. The reaction solution was allowedto warm to −50° C. over 50 min. The product solution was thenpartitioned between aqueous potassium phosphate buffer solution (pH 7.0,0.2 M, 10 mL) and dichloromethane (25 mL). The organic phase wasseparated and the aqueous phase was further extracted with three 15-mLportions of dichloromethane. The organic phases were combined and driedover anhydrous sodium sulfate. The dried solution was filtered and thefiltrate was concentrated, affording a yellow solid. The product waspurified by preparatory HPLC on a Coulter Ultrasphere ODS column [10 μm,250×10 mm, UV detection at 350 nm, Solvent A: water, Solvent B:methanol, injection volume: 500 μL (methanol), gradient elution of85→100% B over 30 min, flow rate: 3.5 mL/min]. Fractions eluting at21-27 min were collected and concentrated, affording the pentacyclicaddition product in diastereomerically pure form (8.6 mg, 76%, a whitesolid).

R_(f) 0.07 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CD₂Cl₂) δ 15.21(s, 1H, enol), 8.63 (d, 1H, J=4.5 Hz, pyr-H), 8.19 (d, 1H, J=7.5 Hz,pyr-H), 7.54-7.43 (m, 5H, ArH), 7.34 (d, 1H, J=4.5, 7.5 Hz, pyr-H), 5.36(d, 1H, J=12.0 Hz, OCHH′Ph), 5.33 (d, 1H, J=12.0 Hz, OCHH′Ph), 4.03 (d,1H, J=10.7 Hz, CHN(CH₃)₂), 3.36-3.31 (m, 1H, CHCH₂CHCHN(CH₃)₂), 3.23(dd, 1H, J=16.3, 5.6 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.99 (dd, 1H, J=16.3,16.3 Hz, CHH′CHCH₂CHCHN(CH₃)₂), 2.63 (ddd, 1H, J=1.6, 4.4, 10.7 Hz,CHCHN(CH₃)₂), 2.54-2.48 (m, 7H, N(CH₃)₂, CHH′CHCHN(CH₃)₂), 2.19 (dd, 1H,J=1.6, 14.5 Hz, CHH′CHCHN(CH₃)₂), 0.87 (s, 9H, TBS), 0.26 (s, 3H, TBS),0.13 (s, 3H, TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ 187.7, 183.5, 182.6,182.2, 167.9, 161.2, 153.4, 137.6, 134.1, 129.2, 129.1, 129.1, 126.8,123.0, 108.7, 106.9, 82.2, 73.0, 61.8, 47.0, 42.1, 41.4, 30.1, 28.4,26.1, 23.2, 19.3, −2.4, −3.5; HRMS (ES) m/z calcd for (C₃₃H₃₉N₃O₆Si+H)⁺602.2686. found 602.2686.

Deprotection:

Palladium black (3.0 mg, 0.028 mmol, 2.6 equiv) was added in one portionto a solution of the purified pentacyclic addition product from theexperiment above (6.5 mg, 0.011 mmol, 1 equiv) in dioxane-methanol (7:2,9.0 mL) at 23° C. An atmosphere of hydrogen was introduced by brieflyevacuating the flask, then flushing with pure hydrogen (1 atm). Theresulting green mixture was stirred at 23° C. for 7 hr, then wasfiltered through a plug of cotton. The filtrate was concentrated,affording a yellow oil (7.0 mg). The residue was dissolved inacetonitrile (4.5 mL), transferred to a polypropylene reaction vessel,and concentrated aqueous hydrofluoric acid solution (48 wt %, 0.5 mL)was added to the resulting solution at 23° C. The reaction mixture washeated to 35° C. for 27 hr. Excess hydrofluoric acid was quenched by theaddition of methoxytrimethylsilane (3.5 mL, 25 mmol). The reactionmixture was concentrated, affording a yellow solid. The product waspurified by preparatory HPLC on a Phenomenex Polymerx DVB column [10 μm,250×10 mm, UV detection at 350 nm, Solvent A: 0.5% trifluoroacetic acidin water, Solvent B: 0.5% trifluoroacetic acid in methanol-acetonitrile(1:1), injection volume: 500 μL (methanol), gradient elution with 0→20%B over 40 min, flow rate: 4 mL/min]. Fractions eluting at 35-45 min werecollected and concentrated to give a yellow oil. The oil was dissolvedin methanolic HCl (1.0 mL, 0.10 M) and concentrated, affording7-aza-10-deoxysancycline hydrochloride as a yellow powder (3.7 mg, 79%).¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 8.79-8.77 (m, 2H, pyr-H) 7.91(dd, 1H, J=6.8, 6.8 Hz, pyr-H), 4.12 (s, 1H, CHN(CH₃)₂), 3.41-3.22 (m,2H, CHH′CHCH₂CHCHN(CH₃)₂, CHCH₂CHCHN(CH₃)₂), 3.11-3.00 (m, 8H,CHH′CHCH₂CHCHN(CH₃)₂, CHCHN(CH₃)₂, N(CH₃)₂), 2.34 (ddd, 1H, J=12.9, 4.4,2.4 Hz, CHH′CHCHN(CH₃)₂), 1.77 (ddd, 1H, J=12.9, 12.9, 12.9 Hz,CHH′CHCHN(CH₃)₂); UV max (0.01 M methanolic HCl), nm 264, 345;[α]_(D)=−154° (c=0.15 in 0.01 M methanolic HCl); HRMS (ES) m/z calcd for(C₂₀H₂₁N₃O₆+H)⁺ 400.1508. found 400.1504.

Synthesis of (−)-10-Deoxysancycline

Cyclization Step:

A solution of n-butyllithium in hexanes (2.65 M, 59 μL, 0.16 mmol, 4.0equiv) was added to a solution of phenyl 2-(bromomethyl)benzoate (45.6mg, 0.157 mmol, 3.97 equiv) and enone 7 (19.0 mg, 0.0394 mmol, 1 equiv)in tetrahydrofuran (1.57 mL) at −100° C. The resulting light-redsolution was allowed to warm to 0° C. over 30 min. The ice-cold productsolution was then partitioned between aqueous potassium phosphate buffersolution (pH 7.0, 0.2 M, 5 mL) and dichloromethane (25 mL). The organicphase was separated and the aqueous phase was further extracted with anadditional 15-mL portion of dichloromethane. The organic phases werecombined and dried over anhydrous sodium sulfate. The dried solution wasfiltered and the filtrate was concentrated, affording a yellow solid.The product was purified by preparatory HPLC on a Coulter UltrasphereODS column [10 μm, 250×10 mm, Solvent A: water, Solvent B: methanol,injection volume: 1.0 mL (methanol), gradient elution with 85→100% Bover 30 min, UV detection at 350 nm, flow rate: 3.5 mL/min]. Fractionseluting at 25-30 min were collected and concentrated, affording thepentacyclic addition product in diastereomerically pure form (19.2 mg,81%, a white solid).

R_(f) 0.46 (3:7 ethyl acetate-hexanes); ¹H NMR (500 MHz, CD₂Cl₂) δ 15.53(s, 1H, enol), 7.94 (d, 1H, J=7.9 Hz, ArH), 7.54-7.28 (m, 8H, ArH,OCH₂ArH), 5.37-5.34 (m, 2H, OCH₂Ph), 4.05 (d, 1H, J=10.7 Hz, CHN(CH₃)₂),3.24-3.18 (m, 1H, CHCH₂CHCHN(CH₃)₂), 2.99 (dd, 1H, J=15.5, 5.6 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.88 (dd, 1H, J=15.5, 15.5 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.61 (dd, 1H, J=4.4, 10.7 Hz, CHCHN(CH₃)₂),2.54-2.44 (m, 7H, N(CH₃)₂, CHH′CHCHN(CH₃)₂), 2.14 (d, 1H, J=14.3 Hz,CHH′CHCHN(CH₃)₂), 0.86 (s, 9H, TBS), 0.25 (s, 3H, TBS), 0.12 (s, 3H,TBS); ¹³C NMR (100 MHz, CD₂Cl₂) δ 187.8, 183.0, 182.8, 182.4, 167.7,141.7, 135.4, 133.4, 130.9, 129.0, 128.9, 128.9, 128.1, 127.5, 126.5,108.5, 106.8, 82.1, 72.8, 61.5, 58.5, 46.9, 41.9, 38.6, 29.0, 25.9,23.1, 19.1, −2.6, −3.7; HRMS (ES) m/z calcd for (C₃₄H₄₀N₃O₆Si+H)⁺601.2734. found 601.2730.

Deprotection:

Concentrated aqueous hydrofluoric acid solution (48 wt %, 1.1 mL) wasadded to a polypropylene reaction vessel containing a solution of thepentacyclic addition product from the experiment above (15.1 mg, 0.0251mmol, 1 equiv) in acetonitrile (10 mL) at 23° C. The resulting solutionwas stirred vigorously at 23° C. for 12 h, then was poured into water(50 mL) containing dipotassium hydrogenphosphate (4.7 g) and the productwas extracted with ethyl acetate (3×25 mL). The organic phases werecombined and dried over anhydrous sodium sulfate. The dried solution wasfiltered and the filtrate was concentrated, affording a yellow solid(12.2 mg, 99%). The residue was dissolved in methanol-dioxane (1:1, 3.0mL) and palladium black (6.5 mg, 0.061 mmol, 2.4 equiv) was added to theresulting solution in one portion. An atmosphere of hydrogen wasintroduced by briefly evacuating the flask, then flushing with purehydrogen (1 atm). The resulting light-yellow mixture was stirred at 23°C. for 20 min, then was filtered through a plug of cotton. The filtratewas concentrated, affording a yellow solid. The product was purified bypreparatory HPLC on a Phenomenex Polymerx DVB column [10 250×10 mm, UVdetection at 350 nm, Solvent A: 0.01 N HCl, Solvent B: acetonitrile,injection volume: 1.0 mL (methanol containing 10 mg oxalic acid),gradient elution with 5→50% B over 30 min, flow rate: 5 mL/min].Fractions eluting at 16-22 min were collected and concentrated,affording 10-deoxysancycline hydrochloride as a white powder (9.1 mg,83%).

¹H NMR (500 MHz, CD₃OD, hydrochloride) δ 7.96 (d, 1H, J=7.3 Hz, ArH)7.51 (dd, 1H, J=7.3, 7.3 Hz, ArH), 7.39 (dd, 1H, J=7.3, 7.3 Hz, ArH),7.30 (d, 1H, J=7.3 Hz, ArH), 4.04 (s, 1H, CHN(CH₃)₂), 3.31-2.99 (m, 8H,CHCH₂CHCHN(CH₃)₂, CHCHN(CH₃)₂, N(CH₃)₂), 2.87 (dd, 1H, J=15.4, 4.3 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.61 (dd, 1H, J=15.4, 15.4 Hz,CHH′CHCH₂CHCHN(CH₃)₂), 2.21 (ddd, J=12.8, 5.0, 2.5 Hz, CHH′CHCHN(CH₃)₂),1.66 (ddd, 1H, J=12.8, 12.8, 12.8 Hz, CHH′CHCHN(CH₃)₂); UV max (0.01 Mmethanolic HCl), nm 264, 348; [α]_(D)=−147° (c=0.15 in 0.01 M methanolicHCl); HRMS (ES) m/z calcd for (C₂₁H₂₂N₂O₆+H)⁺ 399.1556. found 399.1554.

Biological Testing.

Whole-cell antibacterial activity was determined according to methodsrecommended by the NCCLS (National Committee for Clinical LaboratoryStandards. 2002. Methods for dilution antimicrobial susceptibility testsfor bacteria that grow aerobically: approved standard-fifth edition.NCCLS document M100-S12. National Committee for Clinical LaboratoryStandards. Wayne, Pa.; incorporated herein by reference). Test compoundswere dissolved in dimethyl sulfoxide (DMSO) and the resulting solutionswere diluted in water (1:10) to produce stock solutions with a finalconcentration of 256 μg tetracycline analog per mL. In a 96-wellmicrotiter plate, 50-μL aliquots of stock solutions were dilutedserially into cation-adjusted Mueller-Hinton broth (MHB;Becton-Dickinson, Cockeysville, Md.). Test organisms (50 μL aliquots ofsolutions ˜5×10⁻⁵ CFU/mL) were then added to the appropriate wells ofthe microtiter plate. Inoculated plates were incubated aerobically at35° C. for 18-24 h. The MIC was the lowest concentration of compounddetermined to inhibit visible growth. Five Gram-positive and fiveGram-negative bacterial strains were examined in minimum inhibitoryconcentration (MIC) assays. The Gram-positive strains wereStaphylococcus aureus ATCC 29213, Staphylococcus epidermidis ACH-0016,Staphylococcus haemolyticus ACH-0013, Enterococcus faecalis ATCC 700802(a VRE or vancomycin-resistant enterococcus strain), and Staphylococcusaureus ATCC 700699 (carrying the tetM resistance gene). TheGram-negative strains were Pseudomonas aeruginosa ATCC 27853, Klebsiellapneumoniae ATCC 13883, E. coli ATCC 25922, E. coli ACH-0095 (multiplyantibiotic-resistant), and E. coli ATCC 53868::pBR322 (containing aplasmid encoding tetracycline-resistance). These strains are listedagain below, along with certain other details of their origins and knownresistance to antibiotics.

Bacterial Strains

Gram-Positive Organisms:

Staphylococcus aureus ATCC 29213 QC strain for MIC testingStaphylococcus aureus ATCC 700699 Methicillin- andtetracycline-resistant clinical isolate with intermediate resistance tovancomycin Staphylococcus epidermidis ACH-0018 Clinical isolate(Achillion strain collection) Staphylococcus haemolyticus ACH-0013Clinical isolate (Achillion strain collection) Enterococcus faecalisATCC 700802 Vancomycin-resistant clinical isolate Gram-NegativeOrganisms: E. coli ATCC 25922 QC strain for MIC testing E. coli ATCC53868::pBR322 Laboratory strain carrying a plasmid with atetracycline-resistance marker E. coli ACH-0095 Multiply-resistantclinical isolate (Achillion strain collection) Klebsiella pneumoniaeATCC 13883 QC strain for MIC testing Pseudomonas aeruginosa ATCC 27853QC strain for MIC testingATCC=American Type Culture Collection, Manassas, Va.

Example 8—Alternative Routes to Tetracycline Analogs

Many of the studies described above show the generation of thecarbanionic D-ring precursor by metalization of phenyl esters ofo-toluate derivatives. These self-condensation reactions at timesrequired to use of up to 4-5 equivalents of a given D-ring precursor.The presence of an electron-withdrawing substituent on the α-carbongreatly improves the efficiency of metalation and coupling as describedin Example 7 and elsewhere herein. Lithium-halogen exchange of benzylicbromides conducted in situ in the presence of the AB electrophile hasbeen found to provide coupling products where benzylic metalation fails(see Example 7). These benzylic bromides can be prepared with surprisingefficiencies (near quantitative yields) and are surprisingly stable. Thedevelopments may lead to a coupling reaction that could be conductableon a multi-kilo scale. Many different phenyl ester substituents (seebelow) may be used to optimize a coupling reaction.

The optimal group for benzylic metalation, however, may not be the sameas the optimal group for lithium-halogen exchange. In addition, for thelithium-halogen exchange process, besides ester modification, othermetal reagents may be used including, but not limited to, otheralkyllithium reagents (e.g., phenyllithium, mesityllithium), Grignardreagents (e.g., iso-propylmagesium chloride) and zinc-based systems.Barbier-type couplings will be explored using a variety of zero-valentmetals for coupling.

The AB-ring precursors may also be prepared by alternative routes. Thestep-count for the synthesis of most 6-deoxytetracycline analogs is 14from benzoic acid. Eleven of these 14 steps are dedicated to thesynthesis of the AB-ring precursor. Any improvements in the length orefficiency of the route to these AB-ring precursors will have asubstantial impact on the synthesis overall. Alternative syntheses ofthe AB-ring precursor are shown in FIGS. 22A to 22C and 23A to 23C.Among the strategies for alternative A-ring closure sequences areintramolecular Michael additions, palladium-mediated processes, andiminium ion induce closures. Hypervalent iodine reagents may also beused instead of microbial dihydroxylation in the synthesis of theAB-ring precursors as shown in FIGS. 23A to 23C.

Other Embodiments

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

What is claimed is:
 1. A compound of Formula:

or a salt, isomer, or tautomer thereof; wherein: R₁ is hydrogen;halogen; cyclic or acyclic, substituted or unsubstituted, branched orunbranched aliphatic; cyclic or acyclic, substituted or unsubstituted,branched or unbranched heteroaliphatic; acyl; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; —OR_(A);—C(═O)R_(A); —CO₂R_(A); —CN; —SCN; —SR_(A); —SOR_(A); —SO₂R_(A); —NO₂;—N(R_(A))₂; —NHC(O)R_(A); or —C(R_(A))₃; wherein each occurrence ofR_(A) is independently hydrogen, a protecting group, aliphatic,heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio,arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, orheteroarylthio; R₂ is hydrogen; halogen; cyclic or acyclic, substitutedor unsubstituted, branched or unbranched aliphatic; cyclic or acyclic,substituted or unsubstituted, branched or unbranched heteroaliphatic;acyl; substituted or unsubstituted aryl; substituted or unsubstitutedheteroaryl; —OR_(B); —C(═O)R_(B); —CO₂R_(B); —CN; —SCN; —SR_(B);—SOR_(B); —SO₂R_(B); —NO₂; —N(R_(B))₂; —NHC(O)R_(B); or —C(R_(B))₃;wherein each occurrence of R_(B) is independently hydrogen, a protectinggroup, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy,aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino,heteroaryloxy, or heteroarylthio; R₃ is hydrogen; halogen; cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; acyl; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; —OR_(C); —C(═O)R_(C); —CO₂R_(C); —CN; —SCN;—SR_(C); —SOR_(C); —SO₂R_(C); —NO₂; —N(R_(C))₂; —NHC(O)R_(C); or—C(R_(C))₃; wherein each occurrence of R_(C) is independently hydrogen,a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl,alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino,heteroaryloxy, or heteroarylthio; R₄ is hydrogen; halogen; cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; acyl; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; —OR_(D); —C(═O)R_(D); —CO₂R_(D); —CN; —SCN;—SR_(D); —SOR_(D); —SO₂R_(D); —NO₂; —N(R_(D))₂; —NHC(O)R_(D); or—C(R_(D))₃; wherein each occurrence of R_(D) is independently hydrogen,a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl,alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino,heteroaryloxy, or heteroarylthio; R₅ is hydrogen; halogen; cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; acyl; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; —OR_(E); —CN; —SCN; —SR_(E); or —N(R_(E))₂;wherein each occurrence of R_(E) is independently hydrogen, a protectinggroup, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy,aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino,heteroaryloxy, or heteroarylthio; each occurrence of R₇ is independentlyhydrogen; halogen; cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; acyl; substitutedor unsubstituted aryl; substituted or unsubstituted heteroaryl; —OR_(G);—C(═O)R_(G); —CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); —NO₂;—N(R_(G))₂; —NHC(O)R_(G); or —C(R_(G))₃; wherein each occurrence ofR_(G) is independently hydrogen, a protecting group, aliphatic,heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio,arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, orheteroarylthio; each occurrence of P′ is independently hydrogen or aprotecting group; and n is an integer in the range of 0 to 3, inclusive.2. The compound of claim 1, wherein each instance of R₁, R₂, R₃, and R₄is hydrogen.
 3. The compound of claim 1, wherein R₅ is —N(R_(E))₂,wherein each occurrence of R_(E) is independently hydrogen, a protectinggroup, or lower (C₁-C₆) alkyl.
 4. The compound of claim 3, wherein R₅ is—N(CH₃)₂.
 5. The compound of claim 1, wherein each occurrence of R₇ isindependently halogen; cyclic substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted heteroaryl; —OR_(G);—CO₂R_(G); —CN; —SCN; —SR_(G); —SOR_(G); —SO₂R_(G); or —NHC(O)R_(G);wherein each occurrence of R_(G) is independently hydrogen, a protectinggroup, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy,aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino,heteroaryloxy, or heteroarylthio.
 6. The compound of claim 5, wherein atleast one instance of R₇ is —NHC(O)R_(G).
 7. The compound of claim 5,wherein at least one instance of R₇ is halogen.
 8. The compound of claim7, wherein halogen is —F.
 9. The compound of claim 5, wherein at leastone instance of R₇ is —OR_(G).
 10. The compound of claim 5, wherein atleast one instance of R₇ is cyclic substituted or unsubstitutedheteroaliphatic.
 11. The compound of claim 5, wherein one instance of R₇is —OR_(G) and one instance of R₇ is cyclic substituted or unsubstitutedheteroaliphatic.
 12. The compound of claim 5, wherein one instance of R₇is halogen and one instance of R₇ is —NHC(O)R_(G).
 13. The compound ofclaim 12, wherein halogen is —F.
 14. The compound of claim 5, whereinone instance of R₇ is —OR_(G) and one instance of R₇ is halogen.
 15. Thecompound of claim 14, wherein halogen is —F.
 16. The compound of claim1, wherein n is
 0. 17. The compound of claim 1, wherein n is
 1. 18. Thecompound of claim 1, wherein n is
 2. 19. The compound of claim 1,wherein n is
 3. 20. The compound of claim 1, wherein the compound is:

or a salt, isomer, or tautomer thereof; wherein: n is 0, 1, or 2; and Pis hydrogen, lower alkyl group, acyl group, or a protecting group.